Herpesvirus infection and thoracic aortic aneurysm: Synthesis of evidence and contradictions

Abstract

BACKGROUND

Several studies have detected viruses in blood vessel walls of patients with aneurysms; however, the role played by this association in the pathogenesis of thoracic aortic aneurysm (TAA) has not been systematically investigated.

AIM

To evaluate whether herpesvirus infection is associated with TAA.

METHODS

We performed a retrospective study of 43 patients with TAA (25 patients with non-dissecting TAA; 18 patients with dissecting TAA). Aortic tissue samples were analyzed to detect viral proteins [herpes simplex virus type 1, herpes simplex virus type 2, cytomegalovirus, and Epstein-Barr virus (EBV)] by immunohistochemistry or viral DNA by real-time PCR. Inflammatory infiltrates were characterized with CD4 and CD68 antibodies. Descriptive statistics, hypothesis testing, and linear modeling were performed using base R functions.

RESULTS

Immunohistochemical analysis detected no viral proteins in any of the 43 samples. PCR detected EBV DNA in only one patient (1/43, 2.3%), who was in the dissection group (1/18, 5.6%). Inflammatory infiltrates in the dissection group consisted predominantly of CD4⁺ T cells and CD68⁺ macrophages. Statistically significant intergroup differences in neither age nor sex were observed (P = 0.385).

CONCLUSION

The presence of the virus suggests that herpesvirus infection and TAA can coexist. Our findings do not support a causal relationship between herpesvirus infection and TAA in this cohort, and further larger prospective studies need to be conducted.

Key Words: Thoracic aortic aneurysm; Aortic dissection; Herpesvirus infection; Inflammation; Pathogenesis

Core Tip: In this study, Epstein-Barr virus DNA was detected in only one patient with aortic dissection (5.6%). Inflammatory infiltrates in the dissection group consisted predominantly of CD4⁺ T cells and CD68⁺ macrophages. Our findings do not support a causal relationship between herpesvirus infection and thoracic aortic aneurysm in this cohort.

INTRODUCTION

Viruses have been found to contribute to the pathogenesis of many disorders, including cardiovascular diseases. Atherosclerosis and aneurysms are the most common aortopathies. The role played by viral infection in the formation of atherosclerotic arterial lesions has been discussed since the 1970s. The accumulated evidence supporting the pivotal role of inflammation in atherosclerosis made it natural to suggest that viral infection of the vascular wall is a potential trigger of inflammation. Several studies have detected viral DNA in atherosclerotic plaques of the aorta and coronary arteries[1,2]. Viral infection can activate macrophages and T lymphocytes, thus inducing synthesis of signaling molecules and activating several enzymes that disrupt the integrity of the vascular intima[3]. Morphological examination of atherosclerotic plaques has revealed that macrophages, T cells, and mast cells are predominant. HLADRpositive memory cells have been found among T cells; a small percentage of them express the interleukin (IL)-2 receptor. IL-2R expression in atherosclerotic plaques may be a marker of recent inflammatory activity. Several studies have demonstrated that interferongamma (IFN-γ), IL-2, and lymphotoxin predominate in atherosclerotic areas of the vascular wall; these cytokines drive cell-mediated immune reactions to eliminate intracellular pathogens[4]. The current concepts suggest that macrophage activation occurs in the atherosclerotic plaque and involves many factors, including infection. Findings indicate that macrophages synthesize pro-inflammatory cytokines upon viral infection. Thus, macrophages actively synthesize IFN-γ, produce IL-18, and display upregulated expression of major histocompatibility complex (MHC) class II molecules, CD40, CD80, and CD86 in response to cytomegalovirus (CMV) infection[5,6]. Another study has demonstrated that CMV infection leads to immunological dysfunction. The macrophage polarization shift toward the deactivated M2c phenotype is associated with decreased synthesis of pro-inflammatory cytokines, inhibition of MHC class I and II expression, as well as a reduced ability to stimulate CD4+ T-cell activation and proliferation[7]. Furthermore, herpes simplex virus prevents type I interferon production by expressing a viral inhibitory protein impairing STINGIRF activation[8]. The mechanisms via which viruses can block macrophage activation have been described. For example, Epstein-Barr virus (EBV) encodes several lytic proteins that inhibit IFN-γ by activating the JAKSTAT signaling pathways[9]. Therefore, along with activating macrophages and T lymphocytes, viral infection can initiate the synthesis of signaling molecules regulating the inflammatory response.

Hence, the morphological, functional, cellular, and molecular mechanisms of vascular wall inflammation in which viruses play a crucial role can be identified.

Thoracic aortic aneurysm (TAA) is a complex issue in modern cardiac surgery. Aortic aneurysm has a latent course; the onset of the first symptoms usually implies the development of aortic dissection or aortic rupture. The mortality rate for acute TAA dissection is 75%-80%. Therefore, identifying predictors of aneurysm dissection is extremely relevant in cardiac surgery.

The effect of blood flow on the aortic wall results in destruction of cellular and extracellular structures, causing necrotic changes in smooth muscle cells, degradation of the extracellular collagen matrix, and inflammation[10]. Inflammatory cells play a pivotal role in regulating intercellular interactions in aneurysms. Inflammatory infiltrates consist of various cells, T lymphocytes and macrophages playing a critical role. T cells accumulate in the aneurysm wall in large quantities. Activated CD4+ cells lead to aneurysm progression by synthesizing IFN-γ and matrix metalloproteinase-9[11]. Activation of the signal transducer and activator of transcription 4 and Tbet pathways by Th1 cells induces the synthesis of pro-inflammatory cytokines, including IFN-γ and tumor necrosis factoralpha[12]. Furthermore, the upregulated IFN-γ expression by Th1 cells is accompanied by a decrease in Th2cell cytokine synthesis[13]. It has been confirmed that inhibition of the Th2synthesized cytokines IL4 or IL5 reduces aortic remodeling in abdominal aortic aneurysm[14,15]. In turn, these cytokines activate inflammatory cells, including macrophages. Macrophages play a key role in aneurysm pathogenesis. Upon activation, M1 macrophages synthesize pro-inflammatory cytokines, initiating inflammation and tissue damage. Conversely, activated M2 macrophages exhibit anti-inflammatory properties, promoting inflammation resolution and tissue repair[16,17]. Recent findings attest to the imbalance between M1 and M2 macrophages in aortic aneurysm. Inhibition of the Notch and STAT3 signaling pathways alters the M1/M2 ratio and reduces aneurysm formation[18].

Several studies have reported detecting viruses in the vessel wall of patients with aneurysms. However, to our knowledge, this association has not been systematically investigated in the context of the pathogenesis of TAAs. This study reports our findings, which do not support a causal relationship between herpesvirus infection and TAA. The presence of the virus suggests that herpesvirus infection and TAA may coexist.

MATERIALS AND METHODS

Study subjects and baseline characteristics

We performed a retrospective analysis of the clinical and morphological data from 43 patients with TAA who had undergone elective surgery. The study included patients with aneurysm located in the thoracic aorta: The aortic root, ascending aorta, aortic arch, descending thoracic aorta. Aneurysm wall segments were obtained during prosthetic repair. The study was conducted in compliance with the Declaration of Helsinki and approved by the Local Ethics Committee of the Petrovsky National Research Center of Surgery (Approval No. 3).

Patients were divided into two groups based on the presence or absence of TAA dissection, which was diagnosed using imaging methods (echocardiography, computed tomography, and magnetic resonance imaging) at the Aortic Surgery Clinic of the Petrovsky National Research Center of Surgery.

Tissue processing and histological analysis

Aneurysm wall fragments obtained during surgery were immediately fixed in 10% formalin, processed using standard techniques, and embedded in paraffin. Sections were stained with hematoxylin and eosin.

Immunohistochemical (IHC) staining was performed using a BOND3 immunostainer (Leica biosystems) with antibodies against macrophages (CD68), T cells (CD3, CD4, and CD8), Herpes simplex virus types 1 and 2, EBV, and CMV (Cell Marque, United States). Morphometric analysis was performed using a ScanScope instrument (Leica) with the ImageScope software.

Viral DNA detection

For detecting viral DNA, we performed realtime PCR using commercial test kits: The reagent kit for detection of EBV DNA by realtime PCR and the Herpes simplex virus types 1 and 2/CMV HerpesComplex on a DTprime analyzer (DNA-Technology LLC, Russia). Results were interpreted according to the manufacturer's instructions. The artificially synthesized positive control was included in the test kit. The negative control consisted of a sample that had undergone the DNA extraction steps, with the test kit’s negative control added instead of the biological sample.

Statistical analysis

The R programming language in the R studio environment (Version 1.2.5042) was used for statistical analysis. Descriptive statistics, hypothesis testing, and linear modeling were performed using base R functions. Differences were considered significant at P ≤ 0.05.

RESULTS

Table 1 lists the demographic data of patients with TAA. The patients were allocated into two groups based on the presence or absence of aortic dissection. The study included 43 patients: 58% (n = 25) with non-dissecting TAA and 42% (n = 18), with dissecting TAA. Most study subjects were male (77%, n = 33); 23% (n = 10) were female. Aneurysm dissection was identified in 45% of males (15/33) and 30% of females (3/10). In the dissection group, 83% (15/18) were male and 17% (3/18) were female, with no statistically significant difference in dissection incidence between sexes (P = 0.385). The mean age was 53 years (SE = 6.8) for males and 58 years (SE = 7.9) for females (Table 1).

Table 1 Demographic characteristics of patients with thoracic aortic aneurysm, n (%).

Characteristic	Total (n = 43)	Non-dissecting aneurysm (n = 25)	Dissecting aneurysm (n = 18)	P value
Sex
Male	33 (77)	18 (72)	15 (83)
Female	10 (23)	7 (28)	3 (17)
Mean age (years)
Male	53	56	50	aP = 0.012
Female	58	63	45	bP = 0.004

^aP = 0.012 for the intergroup comparison of mean age in males.

^bP = 0.004 for the intergroup comparison of mean age in females.

Histological examination of aortic aneurysm samples revealed massive inflammatory infiltrates extending into the media and adventitia of the blood vessel (Figures 1 and 2). IHC staining of aneurysm sections using antibodies against herpes simplex virus type 1, herpes simplex virus type 2, CMV, and EBV failed to detect any viral proteins. These findings suggested that no viruses were present in the 43 aneurysm samples.

Figure 1 Aortic aneurysm. Leukocyte infiltrates around the vasa vasorum and newly formed vessels in the adventitia and media (hematoxylin and eosin staining × 200).

Figure 2 Immunohistochemical staining of an aortic aneurysm. Large CD68positive macrophages containing numerous lysosomes actively resorb necrotic fibers (cell nuclei are stained with hematoxylin × 200).

PCR amplification of DNA from herpes simplex virus type 1, herpes simplex virus type 2, CMV and EBV yielded the following results. No viral DNA was detected among the 25 tissue samples from non-dissecting aneurysms. Examination of tissue samples from dissecting TAA revealed the presence of EBV DNA in only one of the 18 samples. The virus was detected in a 43yearold male patient with concomitant hypertension and dyslipidemia. No evidence of immunodeficiency was obtained for this patient. Figure 3 shows the fluorescence curve from this patient, with EBV confirmed by realtime PCR. The sensitivity of the realtime PCR assay allowed us to detect at least five copies of EBV DNA.

Figure 3 Fluorescence curves for the Fam channel (Epstein–Barr virus) and Hex channel (PCR internal control). EBV: Epstein-Barr virus; RFU: Relative fluorescence unit.

DISCUSSION

In order to elucidate the potential role of viral infection in the pathogenesis of TAA, we divided patients into two groups: Those with non-dissecting aneurysms and those dissecting aneurysms. The group without dissection was used as a control. Detecting viruses in the dissection group would suggest that a viral infection initiated vascular inflammation, subsequently causing aortic dissection. In our study, patients in group 1 (non-dissecting TAA) displayed only mild inflammatory changes confined to the media. In contrast, patients in group 2 (dissecting TAA) displayed prominent morphological signs of inflammation across all the aortic wall layers.

Taking into account the mild inflammation in patients with non-dissecting aneurysms, factors activating inflammation are likely to drive the transformation from a non-dissecting to a dissecting aneurysm.

The 2023 review[19] proposed a molecular mechanism for the contribution of CMV to the pathogenesis of TAA, noting the fundamental role of 26 mature microRNAs induced by the virus. CMV microRNAs inhibit natural killer cells and cytotoxic T cells, thus eliciting a dysregulated inflammatory response[20,21]. CMV was suggested to play a role in aortic aneurysms: Smooth muscle cells from patients with aortic aneurysms were shown to highly express 5 Lipoxygenase in areas rich in inflammatory infiltrates[22,23]. The 2021 study focusing on abdominal aortic aneurysm[24] reported that CMV infection may stimulate local aortic inflammation, with elevated tumor necrosis factoralpha expression and a trend toward decreased IL-4 production. CMV was detected in patients with aneurysms more frequently than in healthy volunteers. We previously proposed a mechanism for the herpesvirus entry into the aortic wall in TAA[25], suggesting the reflex autonomic arch as a potential route of viral spread to functional aortic zones, detailed in the same publication.

Realtime PCR was used to detect EBV in a patient with dissecting TAA, suggesting that viral infection could have triggered the inflammatory response leading to dissection.

No positive IHC staining in the aortic wall may be caused by the fact that viral proteins are present at very low levels, below the detection threshold. A highly sensitive IHC technique can detect active and indolent CMV infections missed by standard methods[26]. In our study, realtime PCR was the control method. EBV was detected by PCR in one patient (positive control) but not by immunohistochemistry in the same sample. Because large quantities of viral protein detectable by immunohistochemistry are expressed in the lytic cycle, the presence of EBV DNA alone attests to the latent cycle rather than lytic one. A latent infection would preclude viral involvement in aneurysm dissection. Moreover, even if immunohistochemistry had been positive in this single patient, the overall prevalence of the virus in the dissection group would be as low as 5.6% (1/18) and 2.3% (1/43). Those are very low values. We believe that the detected virus indicates that viral infection and TAA can coexist rather than have a causal relationship.

Limitations

In this study, we examined 43 tissue samples of TAA and detected EBV in only one case. No other investigated viruses were found in the remaining samples. The absence of viruses be attributed to the nature of the inflammatory response in the aneurysm wall. Herpesviruses are characterized by latency: The lytic cycle lasts from several hours (alpha-herpesviruses) to several weeks (gamma-herpesviruses)[27]. Therefore, the lack of virus detection could be caused by the latent infection, while the lytic phase—when viruses have initiated inflammation-had occurred earlier, possibly during the pre-hospital stage. Therefore, disease duration and biopsy timing must be taken into account. This issue makes further research highly relevant.

In the patient group with dissecting TAA and no detectable viral infection, morphological examination revealed areas characterized by profound inflammatory reactions. These signs of inflammation suggest that factors other than viruses have triggered the inflammation, leading to aortic dissection.

A major limitation of our study is that there was no control group of patients without aortic aneurysms. Therefore, virus detection may be indicative of the background prevalence of the infection in the general population rather than a causal link to the disease.

Power calculation was not included in this retrospective study. Therefore, a prospective study with a priori power calculation is required for obtaining reliable results characterizing the relationship between herpesvirus infection and TAA.

Another limitation deals with the potential role of infection in atherosclerotic vascular lesions. Research into the viral etiology of atherosclerosisassociated inflammation is ongoing. Although several theoretical mechanisms have been proposed, neither the presence of infection in atherosclerotic plaques nor the associated inflammatory response has unambiguously confirmed a causal relationship yet[28,29].

CONCLUSION

Our findings do not support a causal relationship between herpesvirus infection and TAA in this cohort, and larger prospective studies are needed. The cellular mechanisms of inflammatory activation and intercellular signaling are to be studied meticulously, with careful consideration of disease duration, in order to identify the triggers of inflammation in the aneurysm wall, including ruling out the viral etiology of dissection. A comprehensive approach is essential for advancing the development of novel diagnostic and therapeutic strategies for TAA.