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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1780-1785.)
© 1997 American Heart Association, Inc.

Articles

Cytomegalovirus Infection, Lipoprotein(a), and Hypercoagulability: An Atherogenic Link?

F. Javier Nieto; Paul Sorlie; George W. Comstock; Kenneth Wu; Erwin Adam; Joseph L. Melnick; ; Moyses Szklo

From the Department of Epidemiology, The Johns Hopkins University School of Hygiene and Public Health, Baltimore, Md (F.J.N., G.W.C., M.S.), National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md (P.S.), Division of Hematology, University of Texas Medical School, Houston, Tex (K.W.), and the Division of Molecular Virology, Baylor College of Medicine, Houston, Tex (E.A., J.L.M.).

	Abstract

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Abstract A link between cytomegalovirus (CMV) infection and atherosclerosis has been suggested by experimental, clinical, and epidemiologic studies. We investigated the association between CMV antibody titers in serum collected in 1974 in 300 adult residents in Washington County, Md, and hemostatic parameters in plasma collected in 1987 through 1989, when these individuals participated in the baseline examination of the Atherosclerosis Risk in Communities Study. The cross-sectional association of CMV serum antibodies and hemostatic parameters was also explored in another set of Atherosclerosis Risk in Communities cases and controls. In the longitudinal analyses, CMV titers in 1974 were directly associated with 1987 through 1989 plasma levels of von Willebrand factor, factor VIII, and protein C and negatively associated with activated partial thromboplastin time. In the cross-sectional analyses, CMV titers were directly related to antithrombin III and fibrinogen levels. When the association between CMV antibodies and atherosclerosis was examined in stratified analyses, a significant association was restricted to individuals with high levels of lipoprotein(a) and fibrinogen. These results are compatible with previous evidence suggesting that CMV virus might have procoagulant properties. The possible synergism of CMV infection and resulting hypercoagulability with reduced fibrinolysis due to increased lipoprotein(a) levels deserves further investigation.

Key Words: atherosclerosis • cytomegalovirus • fibrinogen • hemostasis • lipoprotein(a)

	Introduction

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Experimental evidence as well as results from clinical and epidemiologic studies suggests that CMV infection is associated with atherosclerotic disease.^{1 2 3 4} In a recent cohort study among participants in the ARIC Study⁵ who resided in Washington County, Md, high CMV antibody levels in serum samples collected in 1974 were found to be associated with greater carotid intima-media thickness at the time of the ARIC examination (1987 through 1992).

Experimental evidence suggests that CMV infection is associated with a procoagulant state,^{6 7} resulting in changes that could reflect endothelial dysfunction, possibly leading to atherosclerosis. To our knowledge, no epidemiologic study has examined these issues. The main objective of the present report is to describe the association between hemostatic parameters and serum CMV antibodies measured in two sets of samples from the ARIC study previously used for case-control studies of viral infection and atherosclerosis.^{4 5} The association between CMV antibodies and Lp(a) levels was also examined because of recent suggestions of a possible interaction between CMV infection, Lp(a) levels, and impaired fibrinolysis.⁸

	Methods

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Study Population
The two sets of case-control subjects included in this report are subsets of the ARIC Study, a multicenter longitudinal study of natural history and risk factors of clinical and subclinical cardiovascular disease.⁹ The ARIC Study population includes 15 800 participants, initially 45 years to 64 years old, from four US communities (Jackson, Miss; Forsyth County, NC; Minneapolis, Minn; and Washington County, Md) who underwent an extensive interview and clinic examination in 1987 through 1989. This cohort is being reexamined every 3 years.

Longitudinal Sample
Of the 4020 participants from Washington County, Md (one of the ARIC sites), 1410 had also participated in a health survey conducted in the county in 1974 that included a collection of about 15 cc of nonfasting blood.¹⁰ Of these 1410 persons, 949 both had serum collected in 1974 that was available for assay and had completed the first and second ARIC examinations. These individuals form the source population for the selection of 150 cases of carotid atherosclerosis and 150 frequency-matched controls that were included in a nested case-control study reporting on the longitudinal association between viral antibodies and atherosclerosis.⁵ All but two of these 300 participants were white.

As described in detail in a previous report,⁵ case-control status was based on measurements of the IMT obtained by B-mode ultrasound at three carotid artery sites (common, bifurcation, and internal carotid).¹¹ Measurements by trained sonographers were obtained on both sides of the neck. Carotid atherosclerosis was defined based on the average IMT for all measurements taken at the ARIC baseline (first) and follow-up (second) visits. The 150 participants with the highest overall mean IMT were considered cases (mean IMT, 1.04 mm; range, 0.88 mm to 1.59 mm). Controls were the participants with the lowest mean IMT within 10-year age and gender strata, frequency matched to the number of cases in each stratum (mean IMT, 0.62 mm; range, 0.43 mm to 0.74 mm).

Cross-sectional Sample
In addition to the Washington County set of cases and controls, we attempted confirmatory analyses of the longitudinal results on another set of ARIC participants who were included in a previously reported cross-sectional study of herpes virus infections and carotid IMT.⁴ This study included 340 matched case-control pairs selected from among participants from all four ARIC centers and found a modest association between carotid IMT and CMV antibodies measured in serum concurrently collected at the time of ARIC baseline examination. The IMT case-control definition in this study used different cut-point criteria than that used on the longitudinal sample.⁴ In brief, cases had at least two measurements of carotid IMT >2.5 mm or bilateral thickening corresponding to a maximum IMT 1.7 mm in the internal carotid, and/or 1.8 mm in the carotid bifurcation, and/or 1.6 mm in the common carotids. Controls were selected among participants with all IMT measurements below the 75th percentile of the cohort distribution. These additional data also allowed us to examine the cross-sectional association of CMV antibodies and plasma hemostatic factors.

Covariate Information
Information on cigarette smoking, history of diabetes, and educational level was obtained at the baseline ARIC examination (1987 through 1989) by trained interviewers following a standardized protocol.⁹ Medication use was assessed by asking the participants to bring to the clinic all medications taken in the previous 2 weeks. Participants taking lipid-lowering medications were classified as being treated for hypercholesterolemia. Antihypertensive medication use was assessed by asking the participant whether they had taken medication for high blood pressure during the past 2 weeks. Blood pressure measurements and venipuncture were carried out after participants had been fasting for at least 12 hours. Sitting blood pressures were measured with a standardized Hawksley random-zero sphygmomanometer after 5 minutes' rest. Systolic and diastolic blood pressures were defined as the first phase and the fifth phase of Korotkoff sounds. The average of the second and third of three measurements (with 30 seconds' rest between them) is used in this paper.

Laboratory Processing
The serum samples collected in 1974 were frozen at -73°C. In August 1994, the samples were thawed, aliquoted, and shipped with dry ice to the laboratory at the Division of Molecular Virology, Baylor College of Medicine, Houston, Tex. The serum samples collected at the ARIC examination (1987 through 1989) were collected for clinical chemistry analysis and had been thawed once and refrozen at -70°C. They were thawed again for separation into aliquots sent for CMV antibodies analysis to the same laboratory in Houston. CMV antibodies were measured as previously described.^{2 5 12 13} Briefly, solid-phase radioimmunoassay was used for the detection of antibodies using the whole antigen of CMV strain AD169 as capture antigen. Positive-negative values represent the ratio of the average counts of the test serum with CMV antigen to the average counts of the test serum with control antigens from uninfected cells, which is always <2. Titration of serum showed that the positive-negative values were directly related to antibody concentrations.

Lp(a) was measured as the total protein component, which is approximately 30% of the total lipoprotein mass, by using a double-antibody enzyme-linked immunosorbent assay technique for apoprotein(a) detection.¹⁴ Hemostatic variables in plasma collected as part of the baseline (1987 through 1989) examination of the ARIC Study were measured following a protocol previously described.^{15 16 17} In brief, fibrinogen was measured by the thrombin time titration method. Factor VII and factor VIII activities were assessed by determining the ability of the testing sample to correct the clotting time of human factor VII or factor VIII defective plasma. VWF and protein C antigens were determined by enzyme-linked immunosorbent assays. Antithrombin III activity was measured by a chromogenic substrate method. aPTT was measured on an automated coagulometer. White blood cell counts were determined in local hospitals at each of the field centers within 24 hours after venipuncture using automated counters.¹⁸ Previous publications have documented the reliability of these measurements as well as their distribution in the ARIC population.^{18 19 20 21 22}

Statistical Analysis
The comparison of the distribution of study variables in cases and controls has been the subject of previous publications.^{4 5} The present analysis focuses on the relation between CMV antibody levels and hemostatic parameters. Mean levels of hemostatic parameters according to CMV antibody level categories were obtained by analysis of covariance, while adjusting for case-control status, age, gender, and other potential confounding variables. For the longitudinal analyses, CMV antibody levels were categorized using the same three levels previously found related to case-control status in a graded fashion: positive-negative ratio <4; 4-19.9; and 20.⁵ As a result of the lower levels of CMV antibodies in the cross-sectional samples,⁴ the categories for CMV antibodies in the corresponding analyses were established as positive-negative ratio <2; 2-7.9; and 8, which resulted in similar proportional distribution of the study population as that of the longitudinal analyses. Linear trend tests were carried out by testing the statistical significance of the linear regression coefficient for a three-level ordinal variable corresponding to these categories, while adjusting for possible confounders. With the exception of aPTT and protein C, all hemostatic variables were slightly skewed. Thus, all linear regression analyses for hemostatic variables were repeated using log-transformed values with results practically identical to those reported here (not shown). Stratified conditional and unconditional logistic regression analyses were used to investigate whether Lp(a) and hemostatic variables could be effect modifiers of the association between CMV antibodies and atherosclerosis, which we have previously documented.^{4 5} All statistical analyses were conducted using SAS version 6.10 (SAS Institute, Cary, NC).

	Results

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Table 1 shows some descriptive information on the study participants included in both study samples. The age and gender distributions are identical by design. All of the main cardiovascular risk factors were cross-sectionally associated with case-control status.

View this table: [in this window] [in a new window]   Table 1. Characteristics of the Study Population, Washington County, Md (1974, 1987 Through 1992)

As previously reported,⁵ CMV antibody levels in serum samples collected in 1974 were markedly higher than in serum samples collected in 1987 through 1989. Among control subjects from Washington County in the two sets of samples, for example, the median CMV positive-negative values were 8.3 and 3.8, respectively. A total of 36 participants had their CMV antibodies measured in both surveys. With the exception of individuals with low antibody levels in 1974, most of these individuals had substantially lower CMV antibody levels in their second measurement (Fig 1). All quintiles above the median were higher in 1974 than in 1987 through 1989 samples, whereas the quintiles below the median were similar (not shown). The corresponding Pearson correlation was r=.66.

View larger version (30K): [in this window] [in a new window]   Figure 1. CMV positive-negative P-N ratios in 36 Washington County ARIC participants included in both the 1974 and the 1987 through 1989 groups with CMV antibodies measured.

In the longitudinal analyses, after adjustment for age, gender, and case-control status, CMV positive-negative values in 1974 sera were negatively associated with aPTT in plasma collected in 1987 through 1989 (Table 2). However, antibody levels were positively associated with plasma levels of vWF, factor VIII, and protein C. After additional adjustment for variables that have been described as correlates both of viral antibodies and of hemostatic factors (serum cholesterol levels, triglyceride levels, cigarette smoking, and educational level),^{5 23} the estimates in Table 2 remained virtually unchanged (not shown). Other hemo-static parameters such as fibrinogen levels and white blood cell count were not related to CMV antibody levels in the longitudinal analysis (Table 2).

View this table: [in this window] [in a new window]   Table 2. Mean Plasma Hemostatic Parameters in 1987 Through 1989 Plasma1 According to CMV Antibody Titers in 1974

When the cross-sectional relationship between CMV titers and hemostatic parameters was explored, different patterns of association were found (Table 2). The CMV positive-negative categories shown on the right side of Table 2 were constructed so that the proportion of participants in each category was comparable to that in the longitudinal analyses (left side of Table 2). In these analyses, a trend for factor VIII was still present but much weaker, whereas the trends for aPTT, vWF, and protein C were entirely absent. However, these cross-sectional analyses show a direct association between CMV antibodies and antithrombin III and fibrinogen levels. Different categorizations of these cross-sectional CMV positive-negative ratios, as well as further adjustment for potential confounders (serum cholesterol levels, triglyceride levels, cigarette smoking, and educational level), showed essentially the same trends (not shown). There were no statistically significant interactions between any of the parameters shown in Table 2 and case-control status; and when analyses were restricted to control subjects, the associations seen in Table 2 were still evident, although the results were no longer statistically significant due to the smaller sample size (not shown).

The association between CMV antibodies and Lp(a) was also investigated. Although no clear trends on the geometric mean Lp(a) protein levels according to the levels of CMV antibodies shown in Table 2 were observed (results not shown), serum samples that were positive for CMV antibodies in 1987 through 1989 were more likely to have high levels of Lp(a) protein than negative CMV sera. Among CMV positive samples (CMV positive-negative ratio >2), 25.1% had Lp(a) protein levels in the upper quartile for the entire ARIC population [Lp(a) protein 148 µg/mL], as compared with only 14.1% among CMV negative sera (odds ratio, 1.9; 95% confidence intervals, 1.2 to 3.2).

To assess whether Lp(a) and the investigated hemostatic parameters modify the association between CMV antibodies and atherosclerosis described in previous publications,^{4 5} stratified analyses were conducted (Table 3). For both the longitudinal analysis (based on the 1974 CMV antibodies) and the cross-sectional analysis (based on the 1987 through 1989 values), a positive association between CMV antibodies and atherosclerosis was restricted to or was stronger among those with values of Lp(a) protein and fibrinogen above the median levels for the ARIC Study population. In contrast, only weak or no consistent effect modification was observed for factor VII (Table 3) and for the other hemostatic parameters considered in this article (not shown).

View this table: [in this window] [in a new window]   Table 3. Odds Ratios (95% Confidence Intervals) of Carotid Atherosclerosis (Assessed in 1987 Through 1989) in Two Sets of Cases and Controls from the ARIC Study1

	Discussion

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The hypothesis of a causal association between herpesvirus infections and atherosclerosis is supported by experimental evidence in animals and cell cultures,^{24 25 26} pathology studies,^{27 28 29} follow-up studies of transplant patients and patients undergoing angioplasty,^{30 31} and epidemiologic studies.^{1 2 3 4 5} Of the herpesvirus species that infect humans, CMV virus appears to be the one most likely associated with atherosclerotic disease.^{3 32} We recently reported the results from the historical cohort that constitutes one of the study groups for the present report showing that high levels of antibodies against CMV in serum collected in 1974 were associated with thickening of the intima-media layers of the carotid artery measured about 13 years to 18 years later in healthy adult individuals.⁵

Several mechanisms have been proposed to explain these associations.^{3 5} Herpesvirus might be involved in atherogenesis by altering the lipid metabolism of involved cells.^{1 33 34} Viral infection might induce endothelial injury, triggering the development of the atherosclerotic plaque.³⁵ In fact, CMV infection of the endothelium increases the adherence of polymorphonuclear leukocytes, one of the earliest steps in the atherogenesis process.³⁶ Furthermore, the presence of CMV in smooth muscle cells from atherectomy specimens has been shown to be associated with enhanced p53 accumulation in patients developing coronary restenosis after angioplasty.³⁷ Replicating CMV destroys proliferating smooth muscle cells, possibly impairing vessel repair of atherosclerotic lesions.³⁸

Another possible mechanism whereby CMV infection is related to atherosclerotic disease or its manifestations is by inducing hemostatic dysfunction and thrombosis.⁸ In the present study, CMV antibody titers in 1974 were associated with 1987 through 1989 plasma levels of certain hemostatic parameters including factor VIII, vWF, and protein C (Table 2). In addition, there was an inverse association with aPTT, an overall screening test for the intrinsic coagulation pathway. The association of CMV titer with vWF may stem from an increased synthesis due to endothelial dysfunction. The association with factor VIII may reflect that the latter factor exists in plasma as a noncovalent complex with vWF.³⁹ In turn, the apparently paradoxical increased levels of protein C associated with high CMV antibody levels could stem from a compensatory mechanism to counteract the increase in procoagulant factors.

In contrast to the longitudinal results, the cross-sectional analyses showed that the associations above were much less evident or had even disappeared, such as for aPTT, vWF, and protein C. The reasons for this apparent inconsistency are unclear. One possible reason is that a particularly virulent CMV strain affected the Washington County population during the early or mid-1970s and resulted in long-term endothelial damage leading to the observed longitudinal associations. This could also explain the results presented in Fig 1, ie, the remarkably higher CMV titers observed in the 1974 samples as compared with the titers in the 1987 through 1989 samples collected in the individuals who were included in both the longitudinal and the cross-sectional studies. Alternatively, these results could stem from a "saturation effect." Provided that the prevalence of CMV infection increases with age, reaching very high proportions in late adulthood,⁴⁰ it is possible that in 1974 when this cohort was relatively young, there was still some kind of range (real negatives versus positives). This may not have been the case (or less so) later, at the time of the ARIC examination, when the apparent negatives might have been "false-negatives." The concurrent associations of CMV antibodies with fibrinogen and antithrombin III in 1987 through 1989 samples may reflect a more acute reaction to a more recent viral reactivation.

Positive CMV antibodies were associated with Lp(a) protein levels in the present study, only when both variables were categorically defined as binary variables. This result was surprising, given the relatively weak dependence of Lp(a) levels in environmental factors vis-a-vis genetic determinants.⁴¹ Although it has been suggested that Lp(a) may be an acute phase reactant,⁴² the pathophysiologic interpretation of transient increases of Lp(a) following acute myocardial infarction and other disease states remain controversial.⁴³ We failed to observe a significant dose-response trend, however, and thus our finding needs to be replicated in other populations.

Of the hemostatic parameters associated with anti-CMV titers in the studies described in this manuscript, fibrinogen, factor VIII, and vWF have been associated with prevalent coronary disease in the ARIC Study.²³ Other studies have documented an association of incident coronary disease and previous levels of fibrinogen and factor VIII,^{42 43} vWF,^{43 44 45} and protein C.^{43 44 45 46} Of these, only fibrinogen was related to carotid atherosclerosis in the ARIC Study.²³ Lp(a) protein levels also have been found to be associated with carotid atherosclerosis in this population.²²

Overall, these results suggest that CMV infection may be associated with chronic hypercoagulability and are consistent with previous in vitro studies documenting that CMV infection produces a depletion of vWF of cultured endothelial cells.⁶ Other studies have documented the procoagulant properties of CMV and other herpesvirus infections^{47 48 49} and that this phenomenon depends on plasma coagulation factors.⁷ The altered hemostatic parameters could stem from mechanical damage or inflammatory activation of the endothelial lining of the vascular wall due to the viral infection,⁷ changes that could eventually lead to atherosclerosis.⁵⁰ Furthermore, the finding that the association between CMV and atherosclerosis seen in previous reports from our group^{4 5} are practically restricted to individuals with high levels of Lp(a) protein and fibrinogen supports the hypothesis that the prothrombotic properties of CMV may at least partially explain its possible atherogenic effect.⁸ A previous study found that another infectious agent (Chlamydia pneumoniae) was more strongly associated with angiographically-defined coronary artery disease when in combination with high Lp(a) levels.⁵³ The interplay between raised prothrombotic activity, reduced fibrinolytic activity, increased Lp(a) levels, and atherosclerosis has been emphasized recently.⁵¹ The striking homology between Lp(a) and plasminogen may result in Lp(a) binding to fibrin and may block fibrinolysis by preventing activation of plasminogen to plasmin.^{8 41 51} The combination of the procoagulant effects of CMV infection and decreased fibrinolysis may be promoting the development of atherosclerosis.

To our knowledge, this is the first epidemiologic study in apparently healthy adult individuals showing an association of CMV infection and levels of hemostatic parameters and Lp(a), as well as their possible interaction regarding atherogenesis. These findings suggest that hemostatic variables may be a link in the relationship between CMV infection and atherosclerosis, a role that deserves future investigation.

	Selected Abbreviations and Acronyms

 

aPTT = activated partial thromboplastin time ARIC = Atherosclerosis Risk in Communities CMV = cytomegalovirus IMT = intimal medial thickness Lp(a) = lipoprotein(a) VWF = von Willebrand factor

	Acknowledgments

 
The ARIC Study is carried out as a collaborative study supported by National Heart, Lung, and Blood Institute contracts N01-HC-55015, N01-HC-55016, N01-HC-55018, N01-HC-55019, N01-HC-55020, N01-HC-55021, and N01-HC-55022. Dr Comstock is supported in part by career research award HL-21670. The Washington County serum bank is supported by research grant CA 47503 from the National Cancer Institute.

We thank Linda Schramm and Sandra C. Hoffman, MPH, at the Johns Hopkins Training Center for Public Health Research, Hagerstown, Md, for their help in managing and handling the serum samples. The Washington County clinical center, central laboratories, and the ultrasound reading center of the ARIC Cooperative Group, their institutions, coinvestigators, and principal staff who contributed to this report are as follows: The Johns Hopkins University, Baltimore, Md: Carol Shearer, Rita Timmons, Joyce B. Chabot, and Carol Christman; University of Texas Medical School, Houston: Valerie Stinson, Pam Pfile, Hoang Pham, and Teri Trevino; The Methodist Hospital, Atherosclerosis Clinical Laboratory, Houston: Doris J. Epps, Charles E. Rhodes, and Selma M. Soyal; Bowman-Gray School of Medicine, Ultrasound Reading Center, Winston-Salem, NC: Christy Jones, Kathy Joyce, Mary Louise Lauffer, and Suzanne Pillsbury; University of North Carolina, Chapel Hill, Coordinating Center: Ken Kaufman, PhD, Ho Kim, Charmaine M. Marquis, and Alison Meyer.

	Footnotes

 
Reprint requests and correspondence to Dr Nieto, Department of Epidemiology, The Johns Hopkins University, School of Hygiene and Public Health, 615 North Wolfe Street, Baltimore, MD 21205.

Received November 21, 1996; accepted February 24, 1997.

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