Donate Help Contact The AHA Sign In Home
American Heart Association
Arteriosclerosis, Thrombosis, and Vascular Biology
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Arteriosclerosis, Thrombosis, and Vascular Biology. 1998;18:339-348

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nicholson, A. C.
Right arrow Articles by Hajjar, D. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nicholson, A. C.
Right arrow Articles by Hajjar, D. P.
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1998;18:339-348.)
© 1998 American Heart Association, Inc.

Brief Reviews

Herpesviruses in Atherosclerosis and Thrombosis

Etiologic Agents or Ubiquitous Bystanders?

Andrew C. Nicholson; ; David P. Hajjar

From the Departments of Pathology (A.C.N., D.P.H.) and Biochemistry (D.P.H.), Center of Vascular Biology, Cornell University Medical College, New York, NY.

Correspondence to David P. Hajjar, Departments of Pathology and Biochemistry, Center of Vascular Biology, Cornell University Medical College, 1300 York Ave, New York, NY 10021. E-mail dphajjar{at}mail.med.cornell.edu

Abstract

Abstract—The role of herpesvirus infections in the pathogenesis of vascular diseases remains an enigma. Although there is abundant circumstantial evidence of a role for herpesviruses in atherosclerosis and related processes, a cause-and-effect relationship has yet to be definitively established. This article will review the pathological, molecular, and biochemical evidence supporting the hypothesis that herpesviruses are involved in the development of atherosclerosis, restenosis after coronary angioplasty, accelerated atherosclerosis in recipients of heart transplants, and the induction of a prothrombotic phenotype in vascular endothelial cells.


Key Words: herpesviruses • atherosclerosis • thrombosis • endothelium • vascular smooth muscle cell

Atherosclerosis is a complex process involving the interplay of genetic and environmental factors and the involvement of multiple cell types. Because injury to the vascular endothelium is thought to initiate the atherosclerotic lesion, there has been considerable research effort to define the mechanisms responsible for this initial injury. Several risk factors, including hypercholesterolemia, cigarette smoking, hypertension, and diabetes, have been implicated in the development of the disease, but these risk factors are not present in many cases of clinical atherosclerosis. Herpesviruses have been proposed as potential initiators of arterial injury. This theory was based on studies done in the late 1970s when it was shown that an avian herpesvirus could induce atherosclerosis (which resembled the human lesion) in chickens.1 2 3 4 This animal model, coupled with the epidemiological association between herpesviral infection and accelerated atherosclerosis in heart transplant patients and in restenosis after angioplasty, and multiple studies demonstrating herpesviral antigens and nucleic acids in the atherosclerotic vessel wall have led to the hypothesis that herpesviruses initiate or exacerbate human vascular disease processes.5 6 7 8

Eight members of the herpesvirus family are now known to infect humans.9 Infections of HSV-1, HSV-2, and CMV are widespread in the general population and have been the primary candidate viruses whose potential relationship to atherosclerosis has been investigated. HSV types 1 and 2 and CMV can infect human vascular ECs10 11 12 and SMCs.13 14

Epidemiological Evidence Linking Herpesvirus and Atherosclerosis

An association between CMV infection and atherosclerosis was originally based on a case-control study of cardiovascular surgery patients. These patients were compared with a control group of subjects who were not undergoing surgery but who were matched for similar cholesterol levels and other atherosclerosis risk factors.15 In the 157 pairs evaluated, the incidence of positive CMV antibodies was higher in the surgical group than in the control group (90% and 74%, respectively, P<.001). A greater percentage of surgical cases than control subjects had high titers of CMV antibodies (57% and 26%, respectively). There was no correlation between antibody titers and lipid parameters. Similar results were found in a study that compared HSV-1, HSV-2, and CMV antibody status to the results of ultrasonography of the carotid arteries, which are commonly used as a marker of asymptomatic carotid artery thickening.16 Matched case-control pairs (340) from the Atherosclerosis Risk in Communities (ARIC) Study were evaluated. This study found a modest (odds ratio=1.55) though significant (P=.03) positive association between CMV antibodies and asymptomatic carotid artery thickening.

In contrast, a study in a subgroup of Framingham Heart Study patients failed to find an overall association between a history of ever having "fever blisters or cold sores" with either the prevalence or 6-year incidence of coronary heart disease, defined as angina pectoris, coronary insufficiency, or myocardial infarction in patients 58 to 89 years old.17 Previous work in this group of patients had shown a strong correlation of the self-reported history with serological evidence of previous HSV-1 infection. This study does not completely rule out the possibility of such a relationship, particularly since a subgroup of women with recurrent cold sores had a 1.5x relative risk (95% confidence interval, 1.0 to 2.1) of developing coronary heart disease.17

Association of Herpesviruses With Accelerated Atherosclerosis in Recipients of Transplanted Hearts

CAD is a major limitation to long-term survival after cardiac transplantation. Its etiology remains unclear but is thought to involve a chronic, allogeneic, immune response to one or more constituents in the coronary vascular wall. Several studies have suggested a strong correlation between CMV infection and accelerated atherosclerosis in recipients of heart transplants; however, these results remain inconclusive.

At Stanford University, 301 cardiac transplant patients were treated with immunosuppression and were followed up prospectively for the occurrence of CMV infection and the development of atherosclerosis.18 Of those tested, 91 patients developed CMV infections, based on (1) positive cultures for CMV; (2) demonstration of characteristic CMV inclusion bodies in tissue samples; and/or (3) a fourfold rise in IgG CMV antibodies. After 5 years of follow-up, the rate of graft loss due to accelerated atherosclerosis was 69% in CMV-infected patients but only 37% in the non–CMV-infected group.18 Furthermore, there was a 10-fold greater incidence of patients who died with >50% luminal obstruction in their coronary arteries in the CMV-positive compared with the non–CMV-infected patients.18 Similar results were reported from a smaller study at the University of Minnesota.19 This study compared rates of CMV infection and atherosclerosis in 102 immunosuppressed patients who had received a cardiac transplant and had survived for at least 1 year. At 2 years after transplant, 32% of the CMV-positive patients had CAD as opposed to 10% of the CMV-negative patients. These results have been reproduced by investigators from other transplant centers with carefully documented coronary angiography and endomyocardial biopsies.20 Moreover, pathological studies have demonstrated an endothelialitis associated with the accelerated atherosclerosis,21 which was correlated with quantification of the virus in endomyocardial biopsies.22 However, other studies have shown a low frequency of detectable human CMV genome in accelerated CAD as assessed by polymerase chain reaction (PCR)–based techniques and have concluded that CMV was not associated with accelerated transplant CAD.23 24

The mechanism by which CMV infection of vascular cells may enhance the development of accelerated arteriosclerosis in the arteries of transplanted hearts is undetermined. Libby and his colleagues (Salomon et al25 ) have hypothesized that this form of accelerated arteriosclerosis represents a form of chronic immunologic reaction resembling delayed-type hypersensitivity. This localized chronic immune reaction may be initiated by inappropriate expression of histocompatibility antigens (HLA) class II. It is most likely exacerbated by cytokines derived from vascular cells as well as from infiltrating leukocytes. In support of this concept, the coronary artery endothelium can express class II HLA, which may elicit a cellular immune response. Macrophages and T lymphocytes accumulate in transplanted coronary arteries, as would be expected in an ongoing immune or inflammatory response. The contributing role of CMV to this process is unclear. However, it has been shown that CMV infection of cultured ECs upregulates major histocompatibility class II expression,26 major histocompatibility class I expression in human aortic SMCs,27 and enhanced SMC proliferation and intimal thickening of rat aortic allografts.28 In addition, coculture of T lymphocytes with CMV-infected, allogeneic, cultured human ECs results in T-cell activation.29 Finally, peripheral blood mononuclear cells exhibit enhanced lysis of CMV- or HSV-infected vascular SMCs. This could contribute to cell turnover in an active vascular lesion.30

Pathobiological Evidence Linking Herpesvirus and Atherosclerosis

A number of studies have demonstrated the presence of herpesviral nucleic acids, antigens, or particles in normal and atherosclerotic vascular tissue. HSV nucleic acids were demonstrated by in situ hybridization in portions of the ascending aorta removed during coronary bypass surgery.31 Eleven of 160 tissue samples were positive. In most cases, the tissue was histologically normal, ie, nonatherosclerotic; however, in four specimens with intimal thickening, two reacted positively with an HSV-2 probe. The positive staining occurred in cells located in discrete foci of increased cellularity within or adjacent to the intima. CMV antigen was also detected in cells cultured from arterial tissue surgically removed from patients with severe arterial disease.32 In addition, herpesviral virions were identified by electron microscopy in aortic tissue from 10 of 60 patients with atherosclerosis undergoing cardiovascular surgery.33 These viral particles were in various stages of viral replication and included empty nucleocapsids and virions with dense cores. In supportive studies, in situ hybridization techniques were used to show that the frequency of detection of CMV in cultured SMCs from arterial plaques was probably underestimated in previous immunohistochemical staining studies.34 More recently, PCR was employed to detect CMV nucleic acids in 90% of samples obtained from patients with severe atherosclerosis compared with only 53% in patients with minimal or no atherosclerosis.35 The presence of the complete viral genome was demonstrated in these samples by dot blot DNA hybridization and PCR with probes and primers derived from the immediate-early and late-genomic regions. mRNA from the immediate-early but not the late-genomic regions could be demonstrated by in situ DNA hybridization. These findings suggested that CMV may exist in the vessel wall, primarily in a latent state, where expression of immediate-early mRNA occurs36 without expression of mRNA coding for structural capsid proteins. Indeed, CMV DNA is widely distributed throughout the arterial tree,37 lending credence to the possibility that the vascular system may be the site of latency for this virus.

To determine whether viral antigens or nucleic acids in the vessel wall were present at an earlier stage in the formation of the atherosclerotic lesion, immunohistochemical and DNA hybridization studies were performed on coronary arteries and aortas of young trauma victims.38 HSV or CMV was detected in eight of 20 specimens from coronary arteries. The viral DNA or antigens were found in cells in both the intact luminal surface and focal clusters of spindle-shaped or foam cells in the intimal layer, thereby supporting the concept that herpesviruses might participate in the initial stages of the atherosclerotic process.

CMV has also been linked to the pathogenesis of inflammatory abdominal aortic aneurysms.39 40 A PCR-based study detected CMV DNA in 41 aortic lesions excised at surgery and 16 aortic tissues obtained at autopsy. CMV DNA was detected in 88% (7 of 8) of inflammatory aortic lesions with periaortic fibrosis, including 5 of 6 aortic aneurysms, and 61% (20 of 33) of atherosclerotic aneurysms, but only 31% (5 of 16) autopsy samples without inflammation or atherosclerosis. This subendothelial inflammation associated with CMV infection is also observed in human hearts during atherosclerosis of coronary allografts.41

Association of CMV Infection With Restenosis After Angioplasty

Restenosis is a major drawback of percutaneous transluminal coronary angioplasty and occurs in approximately one third to one half of all patients. Restenosis is thought to result from acute mechanical injury to the vessel wall followed by thrombosis and enhanced local gene expression of cytokines, growth factors, growth factor receptors, and cell adhesion molecules by vascular cells and infiltrating inflammatory cells.42 The restenotic lesion is characterized by neointimal hyperplasia due to SMC proliferation and to the synthesis of extracellular matrix.

Epstein and his colleagues (Zhou et al43 ) have recently shown that prior infection with CMV is a strong, independent risk factor for restenosis after coronary atherectomy. Seventy-five patients undergoing coronary atherectomy for CAD were evaluated to determine whether previous exposure to CMV increased their risk of restenosis. Blood levels of anti-CMV antibodies were measured (prior to atherectomy) and correlated with restenosis (determined by coronary angiography 6 months after atherectomy). After 6 months, patients44 seropositive for CMV had a greater reduction in luminal diameter (1.24±0.83 versus 0.68±0.69 mm, P=.003) than did seronegative patients, resulting in a significantly higher rate of restenosis in the seropositive patients (43% versus 8%, P=.002). CMV seropositivity and the CMV titer were independently predictive of restenosis.43

The mechanism(s) by which CMV contributes to restenosis was also addressed by Epstein and his colleagues. They demonstrated that IE84, a CMV protein, binds to and inhibits p53 function.45 Mutations in p53 that lead to loss of function are often accompanied by enhanced stability, which allows the protein to be detectable by immunostaining. They demonstrated that there was a significant concordance between p53 immunoreactivity in human restenotic lesions and the presence of CMV DNA; viral DNA was detected in 11 of 13 (85%) of the p53-immunopositive lesions but in only 3 of 11 (27%) of the p53-immunonegative lesions. Conversely, almost 80% of the CMV-positive lesions (11 of 14) were p53-immunopositive. They speculated that latent CMV could be reactivated after a procedure such as balloon angioplasty, IE84 could inhibit the function of p53, and the inhibition of p53 could contribute to SMC proliferation by blocking p53's inhibition of cell cycle progression.45

The role of CMV/p53 interactions in transplant atherosclerosis is less clear. Research by Baas and colleagues46 has led them to conclude that: "(1) CMV-p53 interactions are not important in the development of accelerated graft arteriosclerosis; or (2) there is an interaction, but it is transient and not detectable at the time points examined in this study; or (3) there is an interaction, but binding of CMV to p53 leads to accelerated degradation of p53, as occurs with HPV-E6."46 Their conclusions were based on immunostaining for the p53 gene product in sections of coronary arteries from 19 transplanted hearts. Staining for p53 was observed in smooth muscle and ECs in only 2 of 19 vessels. CMV nucleic acids had been previously found in 6 of 13 of these hearts by in situ hybridization. However, the p21Cip1/WAF1-mediated p53 growth suppressor pathway (p21Cip/WAF1 encodes a cyclin-kinase inhibitor important in mediating p53-dependent cell-cycle arrest) was determined to be intact, as evidenced by intense staining of p21Cip1/WAF1 in vascular smooth muscle.

Another potential mechanism by which CMV infection of vascular SMCs could increase SMC mass within an atherosclerotic or restenotic lesion would be by inhibiting apoptosis (programmed cell death).47 CMV IE1–and IE2–encoded proteins have been shown to block apoptosis in CMV-infected fibroblasts.48 Whether they do so through an interaction with p53 is unclear. While the protein product of the IE2 gene, IE84, interacts with p53,45 it is likely that IE2 acts to inhibit apoptosis by both p53-dependent and p53-independent pathways.49 Inhibition of apoptosis also occurs in CMV-infected human ECs by a mechanism involving sequestration of p53.44 While uninfected human umbilical vein ECs lost their viability after 48 hours of serum starvation, cell death in hCMV-infected ECs was significantly reduced. Uninfected cells showed typical hallmarks of apoptosis, whereas hCMV-infected cells were resistant to apoptosis. Active replication of hCMV was necessary for the antiapoptotic effect, and p53 was elevated in hCMV-infected cells. In hCMV-infected ECs, p53 was localized in the cytoplasm rather than in the expected nuclear location. The authors suggested that the aberrant subcellular pattern of p53 may have been responsible for the antiapoptotic properties of CMV-infected cells.44 It remains to be determined whether this mechanism occurs in vascular SMCs and contributes to accumulation of these cells in restenotic lesions.

Animal Models of Herpesvirus-Induced Atherosclerosis

The first animal model of herpesvirus-induced atherosclerosis was developed in the late 1970s.1 In this model, pathogen-free, normocholesterolemic chickens were infected with MDV, an avian herpesvirus that causes malignant lymphomas of T-cell origin in chickens50 Uninfected, pathogen-free chickens served as controls. At 32 weeks, the virally infected chickens had grossly visible atherosclerotic lesions in the large coronary arteries, mesenteric and gastric arteries, and major aortic branches (Fig 1Down).1 Histological evaluation of these atherosclerotic lesions demonstrated fibroproliferative lesions with prominent intracellular and extracellular accumulation of lipids, sometimes accompanied by thrombosis. These features are similar to human atherosclerotic lesions (Fig 1Down).4 The atherosclerotic lesions could be prevented by preimmunizing the animals with a related herpes virus of turkeys.3 Feeding the MDV-infected chickens a high-cholesterol diet caused an even greater level of cholesterol accumulation.2 Biochemical analysis of the samples of aortic tissue from the normocholesterolemic, MDV-infected group revealed a significantly higher content of total lipids, particularly free and esterified cholesterol, than in uninfected controls.2 MDV infection increased CE synthesis while reducing CE hydrolase activity, alterations in cellular metabolism that favored arterial lipid accumulation2 (Fig 2Down). Furthermore, the cytoplasmic CE hydrolase could not be activated through the cAMP-dependent protein kinase system after MDV infection.2 These studies demonstrated that herpesvirus infection of an animal, without elevated serum cholesterol levels, could cause the development of a vascular lesion that pathologically and biochemically resembled that of human atherosclerosis.



View larger version (45K):
[in this window]
[in a new window]
  		Figure 1. Histological appearance of MDV-infected, normocholesterolemic chicken arteries at age 8 months. A, Major mesenteric artery. The fatty proliferative lesion is characterized by marked intimal thickening of the artery, abundant foam cells, a fibrous cap, and a lipid core. The endothelium remains intact. The lumen is on the right side of the panel. Weingart–van Gieson's stains, x125. B, Gastric artery. Atheromatous changes include a large number of cholesterol clefts and cellular debris at the base of the intima (lumen is on the right side of the panel). Hematoxylin and eosin stain, x205. C, Coronary artery containing a thrombotic occlusion. Weingart–van Gieson's stains, x150.



View larger version (74K):
[in this window]
[in a new window]
  		Figure 2. Effects of herpesvirus infection on cellular cholesterol (Chol) metabolism. This model depicts the role of herpes- virus infection on vascular SMCs in vascular "foam cell" development. Reduction in lysosomal or acidic CE hydrolase (ACEH) is an early cellular response to HSV infection. In addition, HSV infection can lead to decreased conversion of arachidonic acid (C20:4) to PGI2 and 12-HETE, which in turn can cause a reduction in the intracellular levels of cAMP. Because cAMP activates (via phosphorylation) protein kinase A, there is a subsequent reduction in cytoplasmic or neutral CE hydrolase (NCEH). Reduced hydrolysis of CE resulting from decreased activity of the hydrolytic enzymes contributes to cellular CE accumulation. FFA indicates free fatty acid; ACAT, acyl coenzyme A:cholesterol acyltransferase.

The involvement of CMV has more recently been evaluated in a rat model of accelerated allograft atherosclerosis. SMC proliferation and intimal thickening of rat aortic allografts were increased in recipient rats inoculated with CMV (intraperitoneally) at the time of grafting, in comparison with uninfected rats.28 Infection was associated with inflammation within the allograft and a twofold increase in vascular SMC proliferative rate. In addition, CMV infection was associated with enhanced expression of PDGF-BB and transforming growth factor-ß1 mRNAs.51 In another rat model, the susceptibility of medial and neointimal arterial SMCs to acute CMV infection was investigated in immunocompetent and immunocompromised rats. The carotid artery was injured by balloon catheter in both CMV- and mock-infected animals. It was shown that neointimal cells were more susceptible to infection than medial SMCs. Absence of the endothelium and immunosuppression were necessary for CMV infection, as CMV antigen or DNA was not detected in uninjured arteries or in immunocompetent rats.52 Differences in susceptibility of vascular SMC phenotypes to herpesviral infection are shown by our data, which demonstrate that different lineages of arterial SMCs, from either adult rats or rat pups, exhibit variable HSV-1 infectivity.53

Herpesvirus-Induced Alterations in Vascular Cell Lipid Metabolism

Herpesviral infection has profound effects on cellular cholesterol accumulation. These effects were first noted in feline herpesvirus–infected cell cultures in which there was extensive accumulation of intracellular and extracellular cholesterol crystals.54 To evaluate the mechanisms of altered lipid metabolism after MDV infection,2 avian arterial SMCs in vitro were infected with MDV. This procedure resulted in marked accumulation of cholesterol and CE.55 This specific type of lipid accumulation, which also occurs during formation of the human atheroma, was due in part to decreased CE hydrolysis. Analysis of enzyme activation and kinetics showed that the CE cycle was altered.55 Reduced cytoplasmic cholesteryl esterase activity in lipid-laden, herpesvirus-infected cells was partly due to the inability of the enzyme to be activated by the cAMP protein kinase mechanism, coupled with decreased enzyme synthesis in the infected cell56 (Fig 2Up). These studies were extended to human arterial SMCs infected with HSV-1, which also accumulated saturated CE and triacylglycerols.57 Reduction in the activity of CE hydrolases in infected cells was also impaired due to changes in the physical state of the accumulating CE, which prevented maximal hydrolytic activity.58 Furthermore, there was decreased translation of the RNA that encodes the intracellular CE hydrolases.59 A reduction in PGI2 production57 in virally infected cells further compromised the ability of the infected cell to mobilize cholesterol (Fig 2Up).

Alterations in signal-transduction pathways also affected cholesterol metabolism in HSV-1–infected vascular SMCs. We observed changes in LDL cholesterol binding and internalization, intracellular CE metabolism, and cholesterol efflux after viral infection.60 HSV-1 infection of arterial SMCs enhanced LDL binding and uptake, LDL receptor steady-state mRNA levels, and transcription of the LDL receptor gene. HSV-1 infection also increased CE synthetic and 3-hydroxy-3-methylglutaryl coenzyme A reductase activities, with a concomitant reduction in CE hydrolysis and cholesterol efflux. Viral infection was associated with a time-dependent decrease in protein kinase A activity and an increase in VPK activity commensurate with the accumulation of CE. The relationship between increased VPK activity and alterations in CE accumulation in virally infected cells was explored using an HSV-1 VPK— mutant in which the portion of the HSV-1 genome encoding VPK had been deleted. CE accumulation was significantly increased (>50-fold) in HSV-1–infected cells compared with uninfected cells. The HSV-1 VPK— mutant had no significant effect on CE accumulation.60

The mechanism by which VPK alters cellular host cholesterol trafficking is undefined. Although VPK possesses similarities to eukaryotic protein kinases A and C, the cellular substrates for VPK are unknown. It has been shown that VPK can phosphorylate viral substrates, but it has also been speculated that VPK may phosphorylate some host proteins.61 This is the first demonstration that increased VPK activity is associated with altered activities of kinase-dependent proteins involved in cholesterol trafficking. We have not conclusively demonstrated that VPK specifically phosphorylates serine/threonine residues on those proteins involved in cholesterol metabolism. Because CE accumulation in HSV-1–infected SMCs is abrogated in cells infected with an HSV-1 VPK— mutant, this kinase is clearly implicated in altered lipid metabolism after herpesviral infection.60

CMV can also modulate scavenger receptor activity. CMV infection of human SMCs increases class A scavenger receptor mRNA and modified LDL uptake.62 Cotransfection of an IE72 expression plasmid and a plasmid containing a class A scavenger receptor promoter/reporter gene construct enhanced scavenger receptor promoter activity, implicating IE72 in transcriptional activation of the class A scavenger receptor.62 Taken together, both HSV and CMV infection of vascular cells can result in enhanced uptake and accumulation of lipid.

Viral Induction of Cytokines and Growth Factors

Atherosclerosis has many features in common with other chronic inflammatory processes and wound healing.63 Expression of cytokines and growth factors by vascular and inflammatory cells is thought to contribute to the development of the atherosclerotic or restenotic lesion.42 A number of in vitro studies have demonstrated that herpesviral infection can induce the production and/or release of cytokines or growth factors. These biological response modifiers could further modulate cholesterol metabolism in vascular SMCs or macrophages and contribute to cell proliferation and matrix synthesis. In addition, cytokines could induce the expression of adhesion molecules by the endothelium and thereby effect the ability of the endothelium to bind monocytes or inflammatory cells as discussed below. CMV infection in vitro induces macrophages to increase their expression of RNA for IL-1ß, TNF-{alpha}, and colony stimulating factor-1.64 In a mouse model of herpetic keratitis, there is increased expression of IL-1ß and IL-6.65 HSV-1 infection of monocytes strongly upregulates TNF-{alpha} release.66 CMV infection of monocytes induces release of TNF-{alpha} in vitro and in vivo.67 Although the role of TNF as an antiviral cytokine is unclear, TNF-{alpha} appears to play a role in host defense against CMV in vivo.68 While TNF-{alpha} is an inducer of interferon mRNA transcripts, a well-documented cytokine that has antiherpesviral activity, others have shown that TNF's antiviral activity is not abolished in the presence of antiserum to interferon.69 Alternatively, the antiviral activity of TNF may result from TNF-induced lysis of virally infected cells rather than induction of interferon.70 Paradoxically, TNF has been shown to promote the replication and pathogenicity of rat CMV.71 The immediate-early genes of CMV are capable of specifically activating transcription of the monocyte-macrophage IL-1ß gene, as detected in a chloramphenicol acetyltransferase assay system.72 This result is of interest, in light of the increased expression of IL-1ß in atherosclerotic lesions compared with that of the normal vessel wall.73 74 75 Alternatively, CMV may increase cytokine gene expression by activating specific transcription factors, such as nuclear factor-{kappa}B, activator protein-1, and cAMP-responsive element /B.76 Pseudorabies virus, another enveloped herpesvirus, induces the expression of IL-6 mRNA in infected human fibroblasts.77 CMV upregulates IL-6 mRNA expression and protein release from human ECs.78 Other members of the Herpesviridae family, such as Epstein-Barr virus, contain genes that appear to have important regulatory effects on host cell cytokine synthesis.79 CMV infection also enhances mRNA expression of PDGF and transforming growth factor-ß1 in rat aortic allografts.51 Induction of ROIs may be an underlying mechanism leading to upregulation of gene expression in CMV-infected SMCs. CMV induced intracellular ROI generation within minutes after infection of SMCs. The virus subsequently uses ROIs to enhance its own gene expression and replication. ROIs, through activation of nuclear factor-{kappa}B, can also induce expression of multiple cellular genes.80

Finally, production of growth factors and cytokines within the atherosclerotic lesion may further contribute to lipid accumulation by vascular SMCs and macrophages. For example, both TNF-{alpha} and IL-1ß can modulate LDL receptor–mediated cholesterol metabolism by increasing LDL receptor gene transcription.81 Transforming growth factor-ß182 , PDGF,83 84 and basic fibroblast growth factor85 can upregulate LDL receptor expression in vascular SMCs. However, TNF-{alpha} inhibits macrophage scavenger receptor expression.86 87

Prothrombotic Effects of Herpesviral Infection on the Vascular Endothelium

EC injury is a common feature of viral infection and can alter hemostasis in a direct or indirect manner. Data from our laboratory and others demonstrate that herpesviral infection of ECs in vitro can inhibit anticoagulant functions and induce a procoagulant phenotype. Indirectly, herpesviruses can alter EC function by activation of immune and inflammatory pathways.88

Viral infection can alter hemostatic balance resulting in either hemorrhage or coagulation. Thrombocytopenia is an occasional symptom of common viral infections and is most likely immune system mediated.89 EC infection may represent a common pathway by which viruses alter hemostasis.88 Many human viruses have been shown to infect human ECs in vitro. These include HSV-1, adenovirus type 7, measles virus, parainfluenza type 3, mumps virus, poliovirus type 1, echo-virus type 9, CMV, coxsackievirus B3, and Dengue and Junin viruses.11 90 91 The pathophysiological consequences of EC infection by most of these viruses are unknown.

Herpesviral infection alters the normal thromboresistant surface formed by the intact vascular EC surface by three mechanisms: (1) inhibition of anticoagulant/antithrombotic properties, (2) induction of procoagulant/prothrombotic properties, and (3) indirectly, an increase in binding sites for inflammatory cells, which can further shift the EC surface from thromboresistance by secreting prothrombotic cytokines. Although most experimental evidence demonstrating these effects is derived from in vitro studies using ECs in monolayer culture, there is histological evidence that supports these phenomena in vivo. HSV-1 can induce mucosal lesions that are often accompanied by a local vasculitis with fibrin deposition.92 In addition, disseminated intravascular coagulation is seen in fatal systemic neonatal HSV infections93 and is thought to result in part from direct damage to the vessel wall and exposure of subendothelial collagen.93

Herpesviruses induce a prothrombotic phenotype by inhibiting normal anticoagulant and antithrombotic properties of the endothelium. ECs normally synthesize and express heparan sulfate proteoglycan on their surface. Heparan sulfate proteoglycan is a critical element for recruiting and binding antithrombin III, which is responsible for the inactivation of several coagulation proteases (thrombin and activated coagulation factors IX, X, XI, and XII). HSV infection of the endothelium markedly reduces heparan sulfate proteoglycan synthesis and surface expression by ECs.94 The expression of thrombomodulin on the endothelial surface is also reduced as a result of HSV-1 and HSV-2 infection.95 Because loss of thrombomodulin is concomitant with a reduction in thrombin-dependent protein C activation, inhibition of the protein C/protein S/thrombomodulin pathway is a mechanism that could lead to thrombin generation by reducing the ability of protein C to inactivate factors Va and XIIIa.

The second mechanism by which herpesviruses can alter the vascular endothelium is through the induction of procoagulant/prothrombotic properties. Changes in EC membrane phospholipid topography after herpesviral infection alter the efficiency of assembly of the prothrombinase complex and lead to enhanced thrombin generation.96 HSV-infected ECs, in the presence of purified prothrombin, factor Va, and factor Xa, generated twofold to threefold more thrombin relative to uninfected ECs.96 Furthermore, enhanced thrombin production resulted in increased platelet binding to the HSV-infected endothelium. Thrombin-induced PGI2 production by the infected endothelium was substantially reduced.96 Thrombin-induced PGI2 synthesis and release is a mechanism by which ECs diminish thrombin-induced platelet adherence and aggregation. Therefore, decreased PGI2 synthesis may further enhance platelet adhesion to the HSV-infected endothelium. HSV can also induce transient expression of TF on the EC surface. TF is not normally expressed by ECs but can be induced by endotoxin or cytokines.97 98 TF expression in response to HSV was dependent on the magnitude of infection and increased linearly with increasing multiplicity of infection. Maximal expression (threefold to fourfold greater than in mock-infected cells) was seen at 4 hours, but expression returned to baseline by 20 hours.95 TF mRNA is also transiently induced.99 TF expression did not require replicative infection of HSV-1, since replication-defective virus could also produce TF procoagulant activity.99 CMV also has the capacity to induce procoagulant activity on human ECs, as measured by a reduction in clotting time.100 The cause of this enhanced coagulability may result from prothrombinase complex assembly on the CMV surface.101 Moreover, Pryzdial and Wright101 have shown that assembly of a functional complex between factor Xa and cofactor Va and the ability to form prothrombinase was dependent on the addition of CMV, suggesting that the CMV surface contains the necessary procoagulant phospholipid for coagulation enzyme complex assembly leading to thrombin generation.101

A third mechanism by which herpesvirus infection could contribute to a prothrombotic endothelium is by increasing endothelial binding sites for inflammatory cells and platelets. Subsequent release of inflammatory cytokines by adherent inflammatory cells could potentiate a prothrombotic EC surface. Studies by MacGregor et al10 in the early 1980s showed that human granulocytes had increased adherence to herpesvirus-infected bovine ECs. This increased adherence was not dependent on antibody or complement.10 The mechanism of this increased adherence became clear when it was shown that HSV-infected ECs expressed Fc and C3b receptors, and, in an example of molecular mimicry, viral Gp E was shown to act as an Fc receptor whereas Gp C acted as a complement (C3bi) receptor.102 Visser et al103 subsequently showed that antibodies to HSV further augmented adherence, thus implying that the antibody was acting as a bridge between the viral Gp E (Fc receptor) and the granulocyte.103 Enhanced attachment of granulocytes to the virally infected endothelium resulted in increased granulocyte-mediated lysis of HSV-infected ECs.103 CMV infection of ECs also increased the adherence of granulocytes.104 Increased adherence was abolished by tunicamycin, an agent that prevents glycosylation, suggesting that Gp's play a role in the adhesive event.104 The increased binding of granulocytes could not be induced by supernatants from the CMV-infected cells, thereby implying that adherence is a "cell-bound phenomena and is not induced by an adherence-stimulation factor."104 This result is in contrast to the increased binding of monocytes to the HSV-1–infected endothelium, which can be stimulated by a factor secreted into the medium.105

These data are consistent with our model in which we propose that virally encoded Gp's expressed on the surface of the HSV-infected ECs bind and promote activation of factor X, contribute to thrombin generation, and enhance monocyte adhesion through the induction of specific adhesion molecules (Fig 3Down).106 We have demonstrated that increased adhesion of monocytic cell lines to herpesvirus-infected ECs was blocked by antibodies directed against viral Gp C but not by antibodies to Gp D or Gp E.106 Adhesion was dependent on local generation of thrombin, since it could be inhibited by treatment with specific thrombin inhibitors or by growing the ECs in prothrombin-depleted serum. In addition, labeled factor X bound to murine L cells, which were transfected with the gene for herpesvirus Gp C, but not to the nontransfected cells.106 Cross-linking and immunoprecipitation studies demonstrated that factor X and Gp C formed a complex on the cell surface. In other studies, we used partially overlapping peptides representative of different regions of the catalytic domain of factor X to identify a site in factor X that bound to Gp C.107 These peptides blocked factor X–mediated procoagulant activity and suppressed monocyte adhesion to HSV-infected endothelium.107 These data suggest that the interaction of factor X with Gp C on the surface of HSV-infected ECs leads to formation of factor Xa and thrombin, and that thrombin, by upregulating expression of adhesion molecules, causes enhanced monocyte binding (Fig 3Down). We have subsequently identified a factor Xa receptor expressed on vascular endothelial and SMCs,108 but we have not yet determined the role of this receptor in prothrombinase complex formation in virally infected cells.



View larger version (33K):
[in this window]
[in a new window]
  		Figure 3. Herpesvirus induction of endothelial procoagulant activity. This model illustrates cellular mechanisms by which herpesvirus infection of the vascular endothelium may induce a prothrombotic and inflammatory phenotype. Productive viral infection results in synthesis and expression of viral Gp C (gC) by the endothelium. This viral Gp acts as a binding site for the coagulation protein factor (F) X. Gp C can also serve as an Fc and complement (C3b) receptor. Factor X is proteolytically cleaved at the EC surface to its active form, factor Xa, which binds to its cell surface receptor EPR-1, induces a vascular cell mitogenic response, and also catalyzes conversion of prothrombin (factor II) to thrombin (factor IIa). Thrombin, after ligation of the thrombin receptor, can initiate a signaling cascade that induces surface expression of P-selectin, a monocyte and neutrophil receptor, and vWF, which acts as a platelet receptor. It is proposed that the binding of various inflammatory cells and platelets and the subsequent release of cytokines and inflammatory mediators can further amplify this signaling cascade.

ECs express several leukocyte receptors on their surface, including E-selectin (ELAM-1), intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and P-selectin (GMP140, PADGEM, and CD62), in response to cytokines, phorbol esters, or other agonists.109 E-selectin, ICAM-1, and VCAM-1 are induced in response to cytokines (TNF-{alpha} and IL-1ß) in a process requiring protein synthesis. P-selectin is a cytoplasmic protein found on the membrane of Weibel-Palade bodies of resting ECs.110 After stimulation by thrombin, histamine, or complement proteins, the Weibel-Palade body is rapidly translocated and its membrane becomes incorporated into the plasma membrane of the cell, resulting in surface expression.110 This translocation from a preformed intracellular membrane compartment to the cell surface does not require de novo protein synthesis.110 In addition to P-selectin, Weibel-Palade bodies also contain vWF. EC injury induced by either rickettsial infection111 or endotoxin112 results in release of vWF. In contrast, CMV infection causes the disappearance of vWF from ECs.113

Our laboratory has demonstrated that HSV infection of ECs induces cell surface expression of both P-selectin and vWF.114 115 This results functionally in both increased P-selectin–mediated monocyte adhesion114 and increased vWF-mediated platelet adhesion.115 HSV-induced monocyte adhesion is blocked by antibodies directed against P-selectin but not by anti-ELAM or antibodies to other adhesive leukocyte integrins (eg, LFA-1, MAC-1, and P150,95).114 This suggests that P-selectin is a major receptor for monocytes on the HSV-infected endothelium.106

Other studies have shown that HSV-infected ECs secrete fivefold more vWF than do uninfected cells.115 Secretion of vWF paralleled the course of P-selectin expression, appearing by 4 hours and remaining elevated for as long as 24 hours. Increased platelet adhesion was also observed by 4 hours after infection and was inhibited 90% by anti-vWF antibodies. Interaction of platelets with vWF was also inhibited by antibodies directed against platelet Gp Ib but not by antibodies to Gp IIb/IIIa or Gp IV. Increased platelet adhesion was not observed in the presence of the thrombin inhibitor D-Phe-Pro-Arg chloromethyl ketone. These data imply that thrombin, generated after assembly of the prothrombinase complex on the virally infected endothelium, mobilizes vWF from the Weibel-Palade body to the EC surface, where it acts as a platelet receptor.

In Fig 3Up we describe the following molecular mechanism for viral activation of the coagulation cascade: HSV Gp C acts as a binding site for factor X. Concomitant generation of TF converts bound factor X to factor Xa, which then binds to EPR-1, a vascular cell factor Xa receptor; elicits a mitogenic signal to SMCs; stimulates PDGF production116 ; and forms an active prothrombinase complex leading to generation of thrombin. Thrombin, after binding to the thrombin receptor, can induce expression of P-selectin, a monocyte receptor, and vWF, a platelet receptor. The adhesion of inflammatory cells to the HSV-infected endothelium can further amplify the prothrombotic phenotype of the endothelium through the release of cytokines.97 117

Summary

Herpesviruses have been implicated in the etiology and/or pathogenesis of atherosclerosis on the basis of: (1) evidence of widespread and often systemic infection with these viruses in the general population; (2) the presence of antigens and nucleic acid sequences of herpesviruses in atherosclerotic lesions; (3) the association between CMV infection and accelerated atherosclerosis in cardiac allograft recipients and in restenosis after angioplasty; (4) the demonstration of herpesvirus-induced atherosclerosis in animal models; (5) the profound effects on cholesterol metabolism in vascular SMCs infected with herpesviruses, leading to CE accumulation; (6) loss of anticoagulant properties by vascular ECs and the acquisition of procoagulant properties when infected by herpesviruses in vitro; (7) increased binding of inflammatory cells to herpesvirus-infected ECs; and (8) the ability of herpesviruses to induce expression of growth factors and cytokines by vascular and inflammatory cells. However, despite all of this circumstantial evidence, a cause-and-effect relationship between herpesviruses and human atherosclerosis remains to be definitively proved. It is likely that the controversial role of these ubiquitous viruses will continue because of the dilemma of establishing this cause-and-effect relationship in human vascular disease.

Selected Abbreviations and Acronyms

CAD = coronary artery disease
CE = cholesteryl ester
CMV = cytomegalovirus
EC = endothelial cell
Gp = glycoprotein
h = human
HSV-1, -2 = herpes simplex virus type 1, type 2
IL = interleukin
MDV = Marek's disease virus
PDGF = platelet-derived growth factor
PGI2 = prostacyclin
ROI = reactive oxygen intermediates
SMC = smooth muscle cell
TF = tissue factor
TNF = tumor necrosis factor
VPK = virus-induced protein kinase
vWF = von Willebrand factor

Received August 14, 1997; accepted October 21, 1997.

References

  1. Fabricant CG, Fabricant J, Litrenta M, Minick C. Virus induced atherosclerosis. J Exp Med. 1978;148:335–340.[Abstract/Free Full Text]
  2. Hajjar D, Fabricant C, Minick C, Fabricant J. Virus-induced atherosclerosis: herpesvirus infection alters arterial cholesterol metabolism and accumulation. Am J Pathol. 1986;122:62–70.[Abstract]
  3. Fabricant C, Fabricant J, Minick CR, Litrenta M. Herpes virus induced atherosclerosis in chickens. Fed Proc. 1983;42:2476–2479.[Medline] [Order article via Infotrieve]
  4. Minick CR, Fabricant CG, Fabricant J, Litrenta MM. Atheroarteriosclerosis induced by infection by herpesvirus. Am J Pathol. 1978;96:673–706.[Abstract]
  5. Cunningham M, Pasternak R. The potential role of viruses in the pathogenesis of atherosclerosis. Circulation. 1988;77:964–966.[Free Full Text]
  6. Melnick J, Adam E, DeBakey M. Cytomegalovirus and atherosclerosis. Bioessays. 1995;17:899–903.[Medline] [Order article via Infotrieve]
  7. Hajjar D. Viral pathogenesis of atherosclerosis: impact of molecular mimicry and viral genes. Am J Pathol. 1991;139:1195–1211.[Abstract]
  8. Visser M, Vercellotti G. Herpes simplex virus and atherosclerosis. Eur Heart J. 1993;14(suppl K):39–42
  9. Frenkel N, Schirmer E, Wyatt L, Katsofanas G, Roffman E, Danovich R, June C. Isolation of a new herpesvirus from human CD4+ T cells. Proc Natl Acad Sci U S A. 1990;87:748–752.[Abstract/Free Full Text]
  10. Macgregor RR, Friedman HM, Macarak EJ, Kefalides NA. Virus infection of endothelial cells increases granulocyte adherence. J Clin Invest. 1980;65:1469–1477.
  11. Friedman H, Macarak E, MacGregor R, Wolfe J, Kefalides N. Virus infection of endothelial cells. J Infect Dis. 1981;143:266–273.[Medline] [Order article via Infotrieve]
  12. Ho DD, Rota TR, Andrews CA, Hirsch MS. Replication of cytomegalovirus in endothelial cells. J Infect Dis. 1984;150:956–957.[Medline] [Order article via Infotrieve]
  13. Lashgari M, Friedman H, Kefalides N. Suppression of matrix protein synthesis by herpes simplex virus in bovine smooth muscle cells. Biochem Biophys Res Commun. 1987;143:145–151.[Medline] [Order article via Infotrieve]
  14. Tumilowicz JJ, Gawlik ME, Powell B, Trenton J. Replication of cytomegalovirus in human arterial smooth muscle cells. J Virol. 1985;56:839–845.[Abstract/Free Full Text]
  15. Adam E, Melnick J, Probesfield J, Petrie B, Burek J, Bailey K, McCollum C, DeBakey M. High levels of cytomegalovirus antibody in patients requiring vascular surgery for atherosclerosis. Lancet. 1987;2:291–293.[Medline] [Order article via Infotrieve]
  16. Sorlie PD, Adam E, Melnick SL, Folsom A, Skelton T, Chambless LE, Barnes R, Melnick JL. Cytomegalovirus herpesvirus and carotid atherosclerosis: the ARIC study. J Med Virol. 1994;42:33–37.[Medline] [Order article via Infotrieve]
  17. Havlik R, Blackwelder W, Kaslow R, Castelli W. Unlikely association between clinically apparent herpesvirus infection and coronary incidence at older ages: the Framingham Heart Study. Arteriosclerosis. 1989;9:877–880.[Abstract/Free Full Text]
  18. Grattan M, Moreno-Cabral C, Starnes V, Oyer P, Stinson E, Shumway N. Cytomegalovirus infection is associated with cardiac allograft rejection and atherosclerosis. JAMA. 1989;261:3561–3566.[Abstract]
  19. McDonald K, Rector T, Braunlan E, Coubo S, Olivari M. Association of coronary artery disease in cardiac transplant recipients with cytomegalovirus infection. Am J Pathol. 1989;64:359–362.
  20. Koskinen P, Nieminen M, Krogerus L, Lemstrom K, Mattila S Hayry P. Cytomegalovirus infection and accelerated cardiac allograft vasculopathy in human cardiac allografts. J Heart Lung Transplant. 1993;12:724–729.[Medline] [Order article via Infotrieve]
  21. Paavonen T, Mennander A, Lautenschlager I, Mattila S, Hayry P. Endothelialitis and accelerated atherosclerosis in human heart transplant coronaries. J Heart Lung Transplant. 1993;12:117–122.[Medline] [Order article via Infotrieve]
  22. Koskinen P, Krogerus L, Nieminen M, Mattila S, Hayry P, Lautenschlager I. Quantitation of cytomegalovirus infection-associated histologic findings in endomyocardial biopsies of heart allografts. J Heart Lung Transplant. 1993;12:343–354.[Medline] [Order article via Infotrieve]
  23. Gulizia J, Kandolf R, Kendall T, Thieszen S, Wilson J, Radio S, Costanzo M, Winters G, Miller L, McManus B. Infrequency of cytomegalovirus genome in coronary arteriopathy of human heart allografts. Am J Pathol. 1995;147:461–475.[Abstract]
  24. Skowronski E, Mendoza A, Smith S, Jaski B. Detection of cytomegalovirus in paraffin-embedded postmortem coronary artery specimens of heart transplant recipients by the polymerase chain reaction: implications of cytomegalovirus association with graft atherosclerosis. J Heart Lung Transplant. 1993;12:717–723.[Medline] [Order article via Infotrieve]
  25. Salomon R, Hughes C, Schoen F, Payne D, Pober J, Libby P. Human coronary transplantation-associated arteriosclerosis: evidence for a chronic immune reaction to activated graft endothelial cells. Am J Pathol. 1991;138:791–798.[Abstract]
  26. Ustinov J, Loginov R, Bruggeman C, van der Meide P, Hayry P, Lautenschlager I. Cytomegalovirus induces class II expression in rat heart endothelial cells. J Heart Lung Transplant. 1993;12:644–651.[Medline] [Order article via Infotrieve]
  27. Hosenpud J, Chou S, Wagner W. Cytomegalovirus-induced regulation of major histocompatibility complex class I antigen expression in human aortic smooth muscle cells. Transplantation. 1991;52:896–903.[Medline] [Order article via Infotrieve]
  28. Lemstrom K, Bruning J, Bruggeman C, Lautenschlager I, Hayry P. Cytomegalovirus infection enhances smooth muscle cell proliferation and intimal thickening of rat aortic allografts. J Clin Invest. 1993;92:549–558.
  29. Waldman WJ, Adams PW, Orosz CG, Sedmak D. T lymphocyte activation by cytomegalovirus-infected allogeneic cultured human cells. Transplantation. 1992;54:887–896.[Medline] [Order article via Infotrieve]
  30. Datta SK, Tumilowicz JJ, Trentin JJ. Lysis of human arterial cells infected with herpesviridae by peripheral blood mononuclear cells: implications for atherosclerosis. Viral Immunol. 1993;6:153–160.[Medline] [Order article via Infotrieve]
  31. Benditt E, Barrett T, McDougall J. Viruses in the etiology of atherosclerosis. Proc Natl Acad Sci U S A. 1983;80:6386–6389.[Abstract/Free Full Text]
  32. Melnick J, Dreesman G, McCollum C, Petrie B, Burek J, DeBakey M. Cytomegalovirus antigen within human arterial smooth muscle cells. Lancet. 1983;2:644–646.[Medline] [Order article via Infotrieve]
  33. Gyorkey F, Melnick J, Guinn G, Gyorkey P, DeBakey M. Herpes viridae in endothelial cells of the proximal aorta of atherosclerotic patients. Exp Mol Pathol. 1984;40:328–339.[Medline] [Order article via Infotrieve]
  34. Petrie B, Melnick J, Adam E, Burek J, McCollum C, DeBakey M. Nucleic acid sequence of cytomegalovirus in cells cultured from human arterial tissue. J Infect Dis. 1987;155:158–159.[Medline] [Order article via Infotrieve]
  35. Hendrix M, Salimans M, van Boven C, Bruggeman C. High prevalence of latently present cytomegalovirus in artery walls of patients suffering grade III atherosclerosis. Am J Pathol. 1990;136:23–28.[Abstract]
  36. Stinski M, Thomsen D, Stenberg R, Goldstein L. Organization and expression of immediate early genes of the human cytomegalovirus. J Virol. 1983;46:1–14.[Abstract/Free Full Text]
  37. Hendrix M, Daemen M, Bruggeman C. Cytomegalovirus nucleic acid distribution within the human vascular tree. Am J Pathol. 1991;138:563–567.[Abstract]
  38. Yamashiroya H, Ghosh L, Yang R, Robertson A. Herpesviridae in the coronary arteries and aorta of young trauma victims. Am J Pathol. 1988;130:71–79.[Abstract]
  39. Walker DI, Bloor K, Williams G, Gillie I. Inflammatory aneurisms of the abdominal aorta. Br J Surg. 1972;59:609–614.[Medline] [Order article via Infotrieve]
  40. Tanaka S, Toh Y, Mori R, Komori K, Okadome K, Sugimachi K. Possible role of cytomegalovirus in the pathogenesis of inflammatory aortic diseases: a preliminary report. J Vasc Surg. 1992;16:274–279.[Medline] [Order article via Infotrieve]
  41. Koskinen P, Lemstrom K, Bruggeman C, Lautenschlager I, Hayry P. Acute cytomegalovirus infection induces a subendothelial inflammation (endothelialitis) in the allograft vascular wall: a possible linkage with enhanced allograft arteriosclerosis. Am J Pathol. 1994;144:41–50.[Abstract]
  42. Libby P, Schwartz D, Brogi E, Tanaka H, Clinton S. A cascade model for restenosis: a special case of atherosclerosis progression. Circulation. 1992;86(suppl III):III-47–III-52.
  43. Zhou Y, Leon M, Maclawiw M, Popma J, Yu, Z Finkel T, Epstein S. Association between prior cytomegalovirus infection and the risk of restenosis after coronary atherectomy. N Engl J Med. 1996;335:624–630.[Abstract/Free Full Text]
  44. Kovacs A, Weber M, Burns L, Jacob H, Vercellotti G. Cytoplasmic sequestration of p53 in cytomegalovirus-infected human endothelial cells. Am J Pathol. 1997;149:1531–1539.[Abstract]
  45. Speir E, Modali R, Huang E, Leon M, Shawl F, Finkel T, Epstein S. Potential role of human cytomegalovirus and p53 interaction in coronary atherosclerosis. Science. 1994;265:391–394.[Abstract/Free Full Text]
  46. Baas I, Offenhaus J, El-Deiry W, Wu T, Hutchins G, Kasper E, Baughman K, Baumgartner W, Chiou C, Hayward G, Hruban R. The WAF1-mediated p53 growth-suppressor pathway is intact in the coronary arteries of heart transplant recipients. Hum Pathol. 1997;27:324–329.
  47. Schwartz S, Bennett M. Death by any other name. Am J Pathol. 1995;147:229–234.[Medline] [Order article via Infotrieve]
  48. Zhu H, Shen Y, Shenk T. Human cytomegalovirus IE1 and IE2 proteins block apoptosis. J Virol. 1995;69:7960–7970.[Abstract]
  49. Frazier WA. Thrombospondins. Curr Opin Cell Biol. 1991;792:792–799.
  50. Paterson JC, Cottral GE. Experimental coronary sclerosis, III: lymphomatosis as a cause of coronary sclerosis in chickens. Arch Pathol. 1950;49:669–707.
  51. Lemstrom K, Aho P, Bruggeman C, Hayry P. Cytomegalovirus infection enhances mRNA expression of platelet derived growth factor-BB and transforming growth factor-beta 1 in rat aortic allografts. Arterioscler Thromb. 1994;14:2043–2052.[Abstract/Free Full Text]
  52. Persoons M, Daemen M, Bruning J, Bruggeman C. Active cytomegalovirus infection of arterial smooth muscle cells in immunocompromised rats: a clue to herpes virus associated atherosclerosis. Circ Res. 1994;75:214–220.[Abstract/Free Full Text]
  53. Kaner RJ, Medina J, Nicholson AC, Ursea R, Schwartz SM, Hajjar DP. Developmentally regulated herpesvirus plaque formation in arterial smooth muscle cells. Circ Res. 1993;73:10–14.[Abstract]
  54. Fabricant CG, Krook L, Gillespie J. Virus-induced cholesterol crystals. Science. 1973;181:566–567.[Abstract/Free Full Text]
  55. Hajjar D, Falcone D, Fabricant C, Fabricant J. Altered cholesteryl ester cycle is associated with lipid accumulation in herpesvirus-infected arterial smooth muscle cells. J Biol Chem. 1985;260:6124–6128.[Abstract/Free Full Text]
  56. Hajjar D. Herpesvirus infection prevents activation of cytoplasmic cholesteryl esterase in arterial smooth muscle cells. J Biol Chem. 1986;261:7611–7614.[Abstract/Free Full Text]
  57. Hajjar D, Pomerantz K, Falcone D, Weksler B, Grant A. Herpes simplex virus infection in human arterial cells: implications in arteriosclerosis. J Clin Invest. 1987;80:1317–1321.
  58. Hajjar D, Pomerantz K, Snow J. Analysis of the physical state of cholesteryl esters in arterial smooth muscle-derived foam cells by differential scanning calorimetry. Biochem J. 1990;268:693–697.[Medline] [Order article via Infotrieve]
  59. Hajjar D, Nicholson A, Hajjar K, Sando G, Summers B. Decreased messenger RNA translation in herpesvirus-infected arterial cells: effects on cholesteryl ester hydrolase. Proc Natl Acad Sci U S A. 1989;86:3366–3370.[Abstract/Free Full Text]
  60. Hsu H-Y, Nicholson AC, Pomerantz K, Kaner RJ, Hajjar DP. Altered cholesterol trafficking in herpesvirus-infected arterial cells: evidence for viral protein kinase-mediated cholesterol accumulation. J Biol Chem. 1995;270:19630–19637.[Abstract/Free Full Text]
  61. Purves FC, Longnecker R, Leader D, Roizman B. Herpes simplex virus 1 protein kinase is encoded by open reading frame US3 which is not essential for virus growth in cell culture. J Virol. 1987;61:2896–2901.[Abstract/Free Full Text]
  62. Zhou Y, Guetta E, Yu Z, Finkel T, Epstein S. Human cytomegalovirus increases modified low density lipoprotein uptake and scavenger receptor mRNA expression in vascular smooth muscle cells. J Clin Invest. 1996;98:2129–2138.[Medline] [Order article via Infotrieve]
  63. Munro J, Cotran R. Biology of disease: the pathogenesis of atherosclerosis: atherogenesis and inflammation. Lab Invest. 1988;58:249–261.[Medline] [Order article via Infotrieve]
  64. Dudding L, Haskel S, Clark BD, Auron PE, Sporn S, Huang ES. Cytomegalovirus infection stimulates expression of monocyte associated mediator genes. J Immunol. 1989;143:3343–3352.[Abstract]
  65. Staats HF, Lausch RN. Cytokine expression in vivo during murine herpetic keratitis. J Immunol. 1993;151:277–283.[Abstract]
  66. Gosselin J, Flamand L, D'Addario M, Hiscott J, Menezes J. Infection of peripheral blood mononuclear cells by herpes simplex and Epstein-Barr viruses: differential induction of interleukin 6 and tumor necrosis factor-alpha. J Clin Invest. 1992;89:1849–1856.
  67. Smith PD, Saini SS, Raffeld M, Manischewitz JF, Wahl SM. Cytomegalovirus induction of tumor necrosis factor-alpha by human monocytes and mucosal macrophages. J Clin Invest. 1992;90:1642–1648.
  68. Pavic I, Polic B, Crnkovic I, Lucin P, Jonji S, Koszinowski UH. Participation of endogenous tumor necrosis factor in host resistance to cytomegalovirus infection. J Gen Virol. 1993;74:2215–2223.[Abstract/Free Full Text]
  69. Kohase M, Henriksen-DeStefano D, May T, Vilcek J, Sehgal P. Induction of b2-interferon by tumor necrosis factor: a homeostatic mechanism in the control of cell proliferation. Cell. 1986;45:659–666.[Medline] [Order article via Infotrieve]
  70. Wong GHW, Goeddel DV. Tumor necrosis factors a and b inhibit virus replication and synergize with interferons. Nature. 1986;323:819–822.[Medline] [Order article via Infotrieve]
  71. Haagmans B, Stals F, van der Meide P , Bruggeman C, Horzinek M, Schijns V. Tumor necrosis factor alpha promotes replication and pathogenicity of rat cytomegalovirus. J Virol. 1994;68:2297–2304.[Abstract/Free Full Text]
  72. Iwamoto GK, Monick MM, Clark BD, Auron PE, Stinsky MF, Hunningshake GW. Modulation of interleukin 1 beta gene expression by the immediate early genes of human cytomegalovirus. J Clin Invest. 1990;85:1853–1857.
  73. Wang A, Doyle M, Mark D. Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci U S A. 1989;86:9717–9721.[Abstract/Free Full Text]
  74. Clinton S, Fleet J, Loppnow H, Salomon R, Clark B, Cannon J, Shaw A, Dinarello C, Libby P. Interleukin-1 gene expression in rabbit vascular tissue in vivo. Am J Pathol. 1991;138:1005–1014.[Abstract]
  75. Moyer C, Sajuthi D, Tulli H, Williams J. Synthesis of IL-1 alpha and IL-1 beta by arterial cells in atherosclerosis. Am J Pathol. 1991;138:951–960.[Abstract]
  76. Boldogh I, Fons MP, Albrecht T. Increased levels of sequence specific DNA-binding proteins in human cytomegalovirus-infected cells. Biochem Biophys Res Commun. 1993;197:1505–1510.[Medline] [Order article via Infotrieve]
  77. Sehgal P, Helfgott DC, Santhinam U, Tatter S, Clerick R, Ghrayeb J, May LT. Regulation of the acute phase and immune responses in viral disease enhance expression of the beta 2 interferon hepatocyte stimulating factor interleukin 6 gene in virus infected human fibroblasts. J Exp Med. 1988;167:1951–1956.[Abstract/Free Full Text]
  78. Almeida GD, Porada CD, St Jeor S, Ascensao J. Human cytomegalovirus alters interleukin-6 production by endothelial cells. Blood. 1994;83:370–376.[Abstract/Free Full Text]
  79. Moore KW, Vieira P, Fiorentino DF, Trounstein ML, Khan TA, Mosmann TR. Homology of cytokine synthesis inhibitory factor (IL-10) to the Epstein-Barr virus gene BCRFI. Science. 1990;248:1230–1234.[Abstract/Free Full Text]
  80. Speir E, Shibutani T, Yu Z, Ferrans V, Epstein S. Role of reactive oxygen intermediates in cytomegalovirus gene expression and in the response of human smooth muscle cells to viral infection. Circ Res. 1996;79:1143–1152.[Abstract/Free Full Text]
  81. Stopeck A, Nicholson A, Mancini F, Hajjar D. Cytokine regulation of LDL receptor gene transcription in HEP G2 cells. J Biol Chem. 1993;268:17489–17494.[Abstract/Free Full Text]
  82. Nicholson A, Hajjar D. Transforming growth factor-beta up-regulates low density lipoprotein receptor-mediated cholesterol metabolism in vascular smooth muscle cells. J Biol Chem. 1992;267:25982–25987.[Abstract/Free Full Text]
  83. Chait A, Ross R, Albers J, Bierman E. Platelet-derived growth factor stimulates activity of low density lipoprotein receptors. Proc Natl Acad Sci U S A. 1980;77:4084–4088.[Abstract/Free Full Text]
  84. Roth M, Emmons L, Perruchoud A, Block L. Expressions of the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase genes are stimulated by recombinant platelet-derived growth factor enzymes. Proc Natl Acad Sci U S A. 1991;88:1888–1892.[Abstract/Free Full Text]
  85. Hsu H-Y, Nicholson AC, Hajjar DP. Basic fibroblast growth factor-induced low density lipoprotein receptor transcription and surface expression. J Biol Chem. 1994;269:9213–9220.[Abstract/Free Full Text]
  86. Van Lenten B, Fogelman A. Lipopolysaccharide-induced inhibition of scavenger receptor expression in human monocyte-macrophages is mediated through tumor necrosis factor-alpha. J Immunol. 1992;148:112–116.[Abstract]
  87. Hsu H-Y, Nicholson AC, Hajjar DP. Inhibition of macrophage scavenger receptor expression by TNF is regulated at the transcriptional and post-transcriptional level. J Biol Chem. 1996;271:7767–7773.[Abstract/Free Full Text]
  88. Cosgriff T. Viruses and hemostasis. Rev Infect Dis. 1989;11:5672–5688.
  89. Kelton J, Neame P. Hemorrhagic Complications of Infection in Hemostasis and Thrombosis. Philadelphia, Pa: JB Lippincott; 1987:965–974.
  90. Friedman H, Wolfe J, Kefalides N, Macarak E. Susceptibility of endothelial cells derived from different blood vessels to common viruses. In Vitro Cell Dev Biol.. 1986;22:397–401.[Medline] [Order article via Infotrieve]
  91. Friedman H. Infection of endothelial cells by common human viruses. Rev Infect Dis. 1989;11:S700–S704.
  92. McSorley J, Shapiro L, Brownstein M, Hsu K. Herpes simplex virus and varicella-zoster: comparative histology of 77 cases. Int J Dermatol. 1974;13:69–75.[Medline] [Order article via Infotrieve]
  93. Phinney P, Fligiel S, Bryson Y, Porter D. Necrotizing vasculitis in a case of disseminated neonatal herpes simplex infection. Arch Pathol Lab Med. 1982;106:64–67.[Medline] [Order article via Infotrieve]
  94. Kaner R, Iozzo R, Ziaie Z, Kefalides N. Inhibition of proteoglycan synthesis in human endothelial cells after infection with herpes simplex virus type 1 in vitro. Am J Respir Cell Mol Biol. 1990;2:423–431.
  95. Key N, Vercellotti G, Winkelmann J, Moldow C, Goodman J, Esmon N, Esmon C, Jacob H. Infection of vascular endothelial cells with herpes simplex virus enhances tissue factor activity and reduces thrombomodulin expression. Proc Natl Acad Sci U S A. 1990;87:7095–7097.[Abstract/Free Full Text]
  96. Visser M, Tracy P, Vercellotti G, Goodman J, White J, Jacob H. Enhanced thrombin generation and platelet binding on herpes simplex virus-infected endothelium. Proc Natl Acad Sci U S A. 1988;85:8227–8230.[Abstract/Free Full Text]
  97. Bevilacqua M, Pober J, Wheeler M, Cotran R, Gimbrone M. Interleukin-1 activation of vascular endothelium: effects on procoagulant activity and leukocyte adhesion. Am J Pathol. 1985;121:393–403.
  98. Adams DH, Wyner LR, Karnovsky MJ. Experimental graft arteriosclerosis, 2: immunocytochemical analysis of lesion development. Transplantation. 1993;56:794–799.[Medline] [Order article via Infotrieve]
  99. Key N, Bach R, Vercellotti G, Moldow C. Herpes simplex virus type I does not require productive infection to induce tissue factor in human umbilical vein endothelial cells. Lab Invest. 1993;68:645–651.[Medline] [Order article via Infotrieve]
  100. van Dam-Mieras M, Muller A, van Hinsbergh V, Mullers W, Bomans P, Bruggeman C. The procoagulant response of cytomegalovirus infected endothelial cells. Thromb Haemost. 1992;68:364–370.[Medline] [Order article via Infotrieve]
  101. Pryzdial E, Wright J. Prothrombinase assembly on an enveloped virus: evidence that the cytomegalovirus surface contains procoagulant phospholipid. Blood. 1994;84:3749–3757.[Abstract/Free Full Text]
  102. Cines DB, Lyss AP, Bina M, Corkey R, Kefalides NA. Fc and C3 receptors induced by Herpes simplex virus infection in cultured endothelial cells. J Clin Invest. 1982;69:123–128.