© 2000 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
Chronic non-vascular cytomegalovirus infection: effects on the neointimal response to experimental vascular injury
aWashington Hospital Center, Vascular Biology Laboratory, Cardiovascular Research Institute, GHRB-217, 108 Irving Street, Washington, DC, USA
bCardiovascular Surgery Department, UCLA, School of Medicine, Los Angeles, CA, USA
cPathology Branch, NHLBI, National Institutes of Health, Bethesda, MD 20895, USA
dUS Food and Drug Administration, HFM-576, 1401 Rockville Pike, Rockville, MD 20852-1428, USA
* Corresponding author. Tel.: +1-202-877-2362; fax: +1-202-877-2363 yfz1{at}mhg.edu
Received 1 September 1999; accepted 21 October 1999
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionReferences |
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Objective: Epidemiologic and mechanistic evidence implicates a role for cytomegalovirus (CMV) in atherogenesis. Recently, we demonstrated that CMV has the capacity to causally contribute to atherogenesis; acute infection of rats with rat CMV (RCMV) 1 day after carotid artery injury increased neointimal accumulation. Importantly, in the injured vessel infectious virus could not be detected and viral genome was present only transiently, suggesting that additional mechanisms play a role in the virus-induced exacerbation of the vascular injury response other than the changes caused by direct infection of vessel wall cells. The present investigation was designed to determine whether chronic persistent RCMV infection, more relevant to the clinical situation, also exacerbates the response to injury and, if so, whether similar mechanisms are operative. Methods: Sixty 3-week-old male Spraque–Dawley rats received an i.p. injection of either 106 TCID50 RCMV (Priscott strain) or normal saline. The left carotid artery was balloon-injured 3 months after infection. Rats were killed 6 weeks later. This model produces persistent infection, as demonstrated by presence of infectious virus in the salivary glands at time of sacrifice. Results: The neointima to media (N/M) ratio of the injured vessel was 41% greater in the RCMV-infected than in control rats (1.40±0.48 vs. 0.99±0.45; P=0.003). The aorta never contained infectious RCMV, and exhibited RCMV DNA, detected by PCR, only transiently. The persistent infection of non-vascular tissues was associated with increased serum levels of IL-2, IL-4 and IFN-
. Conclusions: CMV infection of young rats causes persistent infection of non-vascular tissues and increased cytokine levels. The neointimal response to subsequent vascular injury is increased, despite absence of virus from the vessel wall. These findings, as in acute infection following vascular injury, suggest that inflammatory and immune responses to chronic persistent CMV infection contribute to an exaggerated response to vascular injury.
KEYWORDS Arteries; Angioplasty; Cytokines; Infection/inflammation; Restenosis
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Evidence deriving from multiple types of studies have demonstrated an association between various pathogens and atherosclerosis [1–8]. These pathogens have included human cytomegalovirus (HCMV), herpes simples virus types 1 and 2, Chlamydia pneumoniae, H. pylori [1–6], and recently hepatitis A virus [7]. Bacterial flora involved in periodontal disease have also been reported to be associated with atherosclerosis [8]. Moreover, we recently demonstrated in a cell model simulating latent CMV infection, that infection of cells with Chlamydia pneumoniae leads to changes compatible with ‘reactivation’ of the virus, suggesting that infection with multiple pathogens may exert additive or synergistic effects [9].
Our laboratory has been particularly interested in the potential atherogenic role of HCMV. Although studies have demonstrated an association between HCMV with the development of atherosclerosis and of restenosis, or have demonstrated an ability of the virus to cause cellular changes that are consistent with a pro-atherosclerotic or pro-restenotic effect [10–12], none of these studies provide direct evidence indicating that the virus has the capacity to contribute causally to either of these disease processes. We therefore initiated a study [13] employing the standard rat carotid injury model to test the concept of causality. In that study we demonstrated that acute infection with CMV of immunocompetent adult rats with rat CMV (RCMV) immediately after carotid artery injury increased the neointimal response to injury [13]. However, if CMV contributes causally to clinical restenosis, acute infection with virus at the time of angioplasty is an unlikely event. In the present investigation we therefore developed a more clinically relevant model in which injury is produced in a rat with chronic persistent RCMV infection. In addition, in our prior acute infection study infectious virus could not be detected in the vessel wall and viral genome was present only transiently [13]. This suggested additional mechanisms play a role in the virus-induced exacerbation of the vascular injury response other than the changes induced by direct infection of vessel wall cells. The present investigation was therefore designed to answer two questions: (1) whether chronic persistent RCMV infection also exacerbates the response to vascular injury and, if so, (2) whether we could identify mechanisms other than those caused by direct infection of the vessel wall that might contribute to the observed response.
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2 Methods |
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2.1 Virus
Rat cytomegalovirus (RCMV), Priscott strain, was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA), and propagated in Rat-2 cells (rat embryonic fibroblast cell line, ATCC). Rat-2 cells were cultured with Dulbecco's minimal essential medium (DMEM) supplemented with 5% fetal bovine serum (FBS). The rat-2 cell monolayers at 80% confluence were infected with RCMV at a multiplicity of infection (MOI) of 0.1. When 100% of the cells exhibited cytopathic effects (CPE) 48–72 h later, the medium (which contained virus) was collected and centrifuged to remove cell debris. The supernatant was collected and stored as virus stock at –80°C until used. Viral infectivity titrations of the RCMV stock was performed on Rat-2 cells propagated in 96-well microtiter plates. Infectivity was recorded according to the induction of CPE by serial 10-fold dilutions of the sample and expressed as units of TCID50 (50% tissue culture infective doses) assay. Stock virus infectivity was 105 TCID50/0.1 ml.
2.2 Animals
All animals were studied under protocols approved by the Animal Care and Use Committee of the National Heart, Lung, and Blood Institute and in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996). Three-week-old male Sprague–Dawley rats weighing 50–100 g (Zivic-Miller, Zelienople, PA, USA) were used for the experiments. All surgical procedures were performed under general anesthesia and using sterile technique. General anesthesia was administered using ketamine 150 mg/kg and xylazine 15 mg/kg i.m. and supplemental ketamine/xylazine i.p. as necessary.
2.3 Experimental design
Rats at 3 weeks of age were randomly divided into two groups. Thirty-six rats received an i.p. injection of 1 ml rat CMV (106 TCID50/ml), and thirty-six rats received an i.p. injection of 1 ml virus-free culture medium as control. Three months later, standard left carotid balloon injury was performed on sixty rats (thirty from each group had surgery, six from each group were sacrificed for cytokine study) as described by Clowes et al. [14]. Each rat was anesthetized with an i.m. injection of ketamine and xylazine. The distal left common carotid artery was exposed at the bifurcation of the internal and external carotid arteries through a midline incision in the neck. A 5-mm arteriotomy was made in the external carotid and the catheter was introduced in a retrograde fashion to the arch of the aorta. The balloon was inflated with 1.5 ml of normal saline to generate slight resistance and passed three times in the common carotid. The external carotid was then tied off and the wound was closed with 2.0 silk.
2.4 Neointimal/medial ratio determination
The rats of both the infected and noninfected groups were killed 6 weeks after balloon injury. Preparation of the balloon injured left carotid arteries was performed as previously described [13,15]. Briefly, the rats were perfused with 10% formalin for approximately 5 min, after which the left carotid arteries were isolated and excised. Then the arteries were cut into 3–5 segments, paraffin embedded, and Movat stained. The slides were inspected visually to select the segment with greatest luminal narrowing. The slides were coded so that the individual carrying out the measurements was unaware of treatment assignment. The areas of the media and the neointima were assessed using computerized image analysis. Neointimal areas were normalized for vessel size by expressing the results as the ratio of neointimal to medial area (N/M).
2.5 Virus recovery
To determine whether infectious virus was present in the blood vessels after RCMV injection, separate groups of rats were used; three animals were sacrificed at 3 days, 1 week, 3 weeks, and 6 weeks after infection. The aortas of the infected rats were homogenized and the homogenate was used to inoculate cells permissive for RCMV. The presence of infectious virus was determined by whether or not cytopathic effects (due to replicating virus) were observed in the monolayer of permissive cells. The salivary glands of each animal were also analyzed in this way to determine whether these tissues, known to be the site of CMV persistence in mice [16,17], harbored infectious virus under the conditions employed in the present investigation.
2.6 Detection of CMV DNA sequences in salivary gland and aorta
If infectious virus was not present in the aorta of infected rats, the virus might still be present and exert cellular effects through expression of its immediate early gene products in the absence of viral replication. To determine whether RCMV was present in the vessel wall, PCR amplification to detect RCMV DNA sequences in the aorta and salivary gland was performed.
PCR methodology: For detection of viral DNA in these organs, DNA was isolated by standard procedures from frozen tissues. PCR was performed with specific primers for the RCMV exon 4 region. Primer: 5'-CTT GTA ATT GCC ATC AAG ACC GCG-3' and 5'-CTC TCT GGC ATT CGT TAG GAT GAA-3'; DNA PCR kit (Perkin-Elmer Cetus, Norwalk, CT, USA) was used according to the manufacturer's recommended procedures under the following conditions: denaturation, 94°C for 1 min; primer annealing, 65°C for 1 min; and extension, 72°C for 1 min in a thermocycler for two rounds (35 cycles each). Amplified products were transferred to a nylon membrane and detected by hybridization. To confirm the integrity of the DNA samples, PCR was performed on extracted DNA using commercially available primers specific for the rat beta-actin gene (Clontech, Palo Alto, CA, USA). The expected (429 bp) genomic fragment was detected in all samples.
2.7 Assay for serum levels of cytokines
Serum samples were collected from six animals of each group at 3 months after rat CMV infection, and thirty animals of each group at 6 weeks following carotid artery balloon injury (3 months after infection) before sacrifice. All samples were aliquoted and stored at –80°C. Serum cytokine levels of IL-2, IL-4, IL-10, TNF-
and IFN- were determined by standard solid-phase sandwich ELISA kit (Biosource International, Camarillo, CA, USA). Assays were performed in duplicate in 96-well microplates according to the manufacturer's protocol directions. Serum samples were 1:1 diluted with standard diluent buffer. The values were determined by using an automated microplate reader and were fitted to a standard curve ranging from 23.4 to 1500 pg/ml (IL-2, IL-4), 39.0 to 2500 pg/ml (IL-10), 2.3 to 150 pg/ml (TNF-) and 21.8 to 1400 pg/ml (IFN-), respectively.
2.8 Statistical analysis
Data are expressed as mean values with standard deviations. Two-tailed Student's t test was used to assess the statistical significance of the neointimal/medial ratio comparison.
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3 Results |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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3.1 Effects of CMV on the vascular response to injury
As assessed as the neointimal to medial (N/M) ratio, the neointimal thickness of animals latently infected with CMV was significantly greater than that of controls (N/M ratio 1.40±0.48 vs. 0.99±0.45; P=0.003) (Fig. 1A–C). When the effects of CMV were assessed by the change in the vessel luminal area in response to balloon injury, and expressed as percentage stenosis, a similar deleterious response was observed (percentage stenosis 44±13% and 32±14% in infected vs. uninfected rat, respectively, P=0.003) (Fig. 1D).
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3.2 Virus recovery
In RCMV infected animals, infectious virus could not be recovered from any of the aortas of infected rats. However, infectious virus was consistently recovered from salivary glands at all time points except 3 days after infection.
3.3 Detection of CMV DNA
RCMV DNA sequences were found in salivary glands of all RCMV infected animals at all time points (Fig. 2 lane 16, depicts salivary gland sampled at 6 weeks as representative). Although RCMV DNA sequences were also detected in the aortas, they were present only transiently; they were detected in two/three rats at 3 days and all rats at 1 week post infection; RCMV DNA sequences were not found in any rat after 1 week (Fig. 2). It should be recalled that carotid injury was performed 3 months after infection.
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3.4 Cytokine response to CMV infection
Three months after CMV infection six animals from each group were killed before balloon injury for cytokine study. Serum IL-2, IL-4 and INF-
levels were significantly greater in the infected than in the mock infected group (IL-2: 75.6±11.2 vs. 35.6±6.2 pg/ml, P<0.001; IL-4: 48.4±8.1 vs. 33.2±5.6 pg/ml, P<0.05 and INF-: 118.7±15.3 vs. 85.2±12.6 pg/ml, P<0.05). There were no statistically significant differences between the infected and the mock-infected groups in the levels of IL-10 or TNF. Six weeks following balloon injury (when the animals were killed for analysis of the injury response) the cytokine levels of thirty animals from each group were similar to the levels measured before injury, 3 months after infection (Fig. 3).
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4 Discussion |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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The results of the present investigation confirm and extend to a more clinically relevant model the results of a previous study performed in our laboratory [13]. The previous study demonstrated that acute infection with CMV, induced the day following balloon injury of the rat carotid artery, caused an increase in the neointimal response to vascular injury. These results demonstrated that CMV infection can augment the vascular response to injury. Because this response is involved in the development of restenosis and of atherosclerosis, the results suggested that CMV may causally contribute to these two pathologic entities. However, if CMV does play a causal role, the clinical model of CMV-induced vascular disease would most likely be based on a chronic, persistent infection produced by the virus.
In immunocompetent individuals, CMV infection usually has no clinically overt sequellae [18]. Instead, like other herpes viruses, CMV resides in host cells chronically, but in a clinically quiescent state [18]. It is not known whether the virus is totally latent (with no expression of viral genes), is producing an abortive infection (expresses immediate early gene products but not late gene products and therefore not replicating), or is replicating but at a sufficiently low rate so as to be clinically silent. The virus could also exist in all three states during the life of the host, undergoing periodic reactivation from latency to produce an abortive infection and/or to intermittently undergo replication. Whatever the precise state of the virus, once infected the host appears to harbor CMV for life [19]. The hypothesis that we have been exploring is whether, in this context of chronic infection of an immunocompetent host, CMV can causally contribute to vascular disease.
The present investigation explores this important issue by using a model of infection more clinically relevant than we used to demonstrate proof of concept. In the current study we infected rats at 3 weeks of age and then performed arterial injury 3 months later. At this time the rats appeared perfectly healthy — however, infectious virus was present, as demonstrated by analysis of the salivary glands, a known site of viral persistence [16,17]. Thus, vascular injury was performed in the context of chronic, persistent CMV infection. Under these circumstances, as with acute infection shortly after vascular injury, CMV infection caused an augmented neointimal response to injury.
Another important finding of this investigation is that, as was observed with the study employing acute infection [13], the CMV-induced vascular effects appeared to derive from mechanisms other than the direct effects of the virus infecting cells residing in the vessel wall. We could not grow virus from the vessel wall of infected animals. We could, however, demonstrate CMV DNA in the vessel wall by PCR amplification, but only transiently; DNA was consistently detected 3 days and 1 week after infection, but could not be detected thereafter. Although we cannot rule out the possibility that early viral presence led to a brief abortive infection that contributed to the exaggerated neointimal response, this appears unlikely in that the virus was no longer present in the vessel wall by the third week post infection, while vessel injury was carried out in the twelfth week.
We should also note that we assayed the aortas instead of the injured carotids for RCMV DNA because the injured carotid arteries were used for histologic analysis and quantitation of lesion size. However, while we believe it is unlikely that viral DNA resided in the injured carotid artery and not the aorta, we cannot rule out this possibility. What makes this unlikely is that in our prior study in which we infected the rats immediately after carotid injury, RCMV DNA could not be detected in the injured carotid after 3 days from infection. These results are therefore compatible with our concept that in addition to possible direct effects of infection on the cells of the vessel wall, chronic persistent CMV infection triggers inflammatory and immune responses (with their associated cytokine responses) that could influence the response of the vessel to injury in a way that would favor the development of restenosis or atherosclerosis [20–22].
The finding of elevated serum levels of IFN, IL-2 and IL-4 following RCMV infection support this concept. IL-2 is a strong mitogen for T lymphocytes, and IFN- activates macrophages to produce cytokines, reactive oxygen species and metalloproteinases, and to stimulate endothelial cells to express adhesion molecules [23,24]. All these pathophysiological changes are involved in the development of atherosclerosis. Although unproven, it is likely that such increases in cytokine levels could also influence the development of neointima in response to the acute vascular injury used in the present study. However, we did not carry out the studies to prove the validity of this conjecture. We can nonetheless conclude, given the chronically elevated cytokine levels produced by infection, that chronic persistent CMV infection of non-vascular tissues produces the type of long-lasting systemic effects that could influence the vascular response to injury; whether such a causal role can be ascribed to these specific changes needs further investigation.
Time for primary review 26 days.
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