Cytomegalovirus infection is associated with an increase in aortic stiffness in older men which may be mediated in part by CD4 memory T-cells.

Human Cytomegalovirus (CMV) infection is associated with atherosclerosis, higher cardiovascular disease (CVD) risk, and an increase in memory T-cells (Tmem). T-cells have also been implicated in CVD, independently of CMV infection. To better understand the CMV-associated CVD risk, we examined the association between CMV (IgG) serostatus and central aortic (carotid-to-femoral) pulse wave velocity (cfPWV), an early, independent predictor of CVD. We also investigated if such an association might be reflected by the distribution of Tmem and/or other T-cell subsets. Methods: Healthy older volunteers (60-93 years) underwent routine clinical and laboratory evaluation, including assessment of cfPWV in eligible participants. Flow-cytometry was used to assess proportions of memory T-cells, CD28null T-cells, and CMV-specific T-cells. The following associations were examined; CMV serostatus/cfPWV, CMV serostatus/proportion of Tmem, proportion of Tmem/cfPWV, CD28null T-cells/cfPWV, and CMV-specific T-cells/cfPWV. Linear regression models were used to adjust for age, sex, socioeconomic status, smoking, waist-to-hip ratio, cholesterol, and blood pressure as required. Results: Statistically significant positive associations were found (P-values for the fully adjusted models are given); CMV serostatus/cfPWV in men (P ≤ 0.01) but not in women, CMV serostatus/proportions of CD4 Tmem in men (P ≤ 0.05) but not in women; proportions of CD4 Tmem/cfPWV among CMV seropositive (CMV+) people (P ≤ 0.05) but not CMV seronegative (CMV-) people. Conclusion: CMV infection increases the CVD risk of older men by increasing cfPWV. This may be mediated in part by increased proportions of CD4 Tmem, higher numbers of which are found in CMV+ older people and more so among men than women. Given the high prevalence of CMV worldwide, our findings point to a significant global health issue. Novel strategies to mitigate the increased CVD risk associated with CMV may be required.

Induction of the Coxsackievirus and Adenovirus Receptor in Macrophages During the Formation of Atherosclerotic Plaques.

Multiple viruses are implicated in atherosclerosis, but the mechanisms by which they infect cells and contribute to plaque formation in arterial walls are not well understood. Based on reports showing the presence of enterovirus in atherosclerotic plaques we hypothesized that the coxsackievirus and adenovirus receptor (CXADR/CAR), although absent in normal arteries, could be induced during plaque formation. Large-scale microarray and mass spectrometric analyses revealed significant up-regulation of CXADR messenger RNA and protein levels in plaque-invested carotid arteries compared with control arteries. Macrophages were identified as a previously unknown cellular source of CXADR in human plaques and plaques from Ldr-/-Apob100/100 mice. CXADR was specifically associated with M1-polarized macrophages and foam cells and was experimentally induced during macrophage differentiation. Furthermore, it was significantly correlated with receptors for other viruses linked to atherosclerosis. The results show that CXADR is induced in macrophages during plaque formation, suggesting a mechanism by which enterovirus infect cells in atherosclerotic plaques.

Human cytomegalovirus infection is correlated with atherosclerotic plaque vulnerability in carotid artery.

BACKGROUND: Several studies have suggested that human cytomegalovirus (CMV) infection is closely related to the pathogenesis of atherosclerosis. The present study aimed to investigate the association between human CMV infection and carotid atherosclerotic plaque vulnerability in a Chinese population. METHODS: In total, 42 patients with carotid atherosclerosis (observation group) and 30 healthy volunteers (control group) were recruited in our study from October 2016 to January 2018. Statistical analysis was carried out to calculate the infection rate of CMV in subjects. Spearman's rank analysis was performed to evaluate the correlation between CMV infection and atherosclerotic plaque vulnerability. RESULTS: The positive rate of CMV was significantly higher in the observation group compared to the control group, and matrix metalloproteinase 9 (MMP-9), tumor necrosis factor-α (TNF-α) and lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) expression levels were also elevated in the observation group compared to those in the control group. In carotid atherosclerotic patients, the detection rate of unstable plaques and the Crouse scores in vulnerable plaque were significantly higher in the CMV-positive group compared to those in the CMV-negative group. As revealed by correlation analysis, CMV infection was significantly positively correlated with plaque vulnerability and expression levels of MMP-9, TNF-α and LOX-1 in carotid atherosclerotic patients. CONCLUSIONS: Human CMV infection might be a potential risk factor for increased plaque vulnerability in patients with carotid atherosclerosis.

Recurrent strokes, central nervous system vasculitis, and acquired protein S deficiency secondary to varicella zoster in a child with AIDS.

A child with vertical transmission of human immunodeficiency virus refractory to therapy developed zoster-induced protein S deficiency and recurrent strokes. Extensive carotid arteritis was found postmortem. The carotid tissue was positive for herpes varicella zoster by polymerase chain reaction, as were immunofixation stains of the arterial wall.

Relationship between brain large artery characteristics and their downstream arterioles.

We aimed to test the hypothesis that brain large artery diameters relate to distal downstream arteriolar diameters. In a sample of 110 autopsied individuals (69% men, 76% HIV+, mean age 51), we used multilevel models to relate large artery lumen and lumen-to-wall ratio to left frontal lobe arteriolar lumen and lumen-to-wall ratio adjusting for demographics and vascular risk factors. Comparing the large artery characteristics of the whole brain did not disclose significant associations with frontal lobe arteriolar characteristics. However, restricting the comparison to large arteries upstream of the studied arterioles demonstrated an independent association between left-sided frontal lobe arteriolar luminal diameter with large artery luminal diameters (B = 1.82 ± 0.77, P = 0.01) and with large artery lumen-to-wall ratio (B = 0.58 ± 0.29, P = 0.05). In stratified models, the point estimates in the HIV+ subsample were larger than in the HIV- subsample. These finding suggest coupling between higher proximal blood flow represented by large artery diameter and lower distal resistance represented by arteriolar dilatation. The relationship between arteriolar dilatation and brain parenchyma homeostasis should be further studied.

Epizootic haemorrhagic disease virus induced apoptosis in bovine carotid artery endothelium is p53 independent.

Epizootic haemorrhagic disease virus (EHDV) replicates in endothelium and it has been shown that EHDV serotype 2 (Ibaraki) is able to cause cell death by apoptosis in cow pulmonary artery endothelial cells. However, the underlying mechanism has not been established. For some viruses, such as influenza, a p53 dependent mechanism has been demonstrated in viral induced apoptosis. In this study, we investigate the involvement of p53 in the induction of apoptosis in a US isolate of EHDV serotype 2 in cow endothelium. We inoculated cow carotid artery endothelial cell cultures with live and inactivated EHDV­2 isolated from a white­tailed deer (Odocoileus virginianus). Using in situ nick end­labeling (TUNEL), caspase­3 (cleaved) immunohistochemistry (IHC), flow cytometry and annexin staining we documented the development of apoptosis and its direct relation to viral replication. p53 gene regulation and protein expression were assessed by reverse transcription polymerase chain reaction and IHC, respectively, in infected cells. We show that p53 mRNA was not upregulated and protein expression was not significantly increased. No increase of p53 mRNA or protein expression was observed in cells that stained positive for EHDV antigen. Our results indicate that EHDV induces apoptosis through a p53 independent mechanism.

Anti-cytomegalovirus antibody levels are associated with carotid atherosclerosis and inflammatory cytokine production in elderly Koreans.

BACKGROUND: Recent studies have implicated human cytomegalovirus (HCMV) infection as a possible etiological factor in cardiovascular disease. We assessed whether anti-HCMV antibody levels are associated with carotid atherosclerosis and inflammatory cytokine production in elderly Koreans. METHODS: Participants (age, ≥65 years) were prospectively enrolled from September 2012 to July 2013 at a 2000-bed university hospital. During the study period, 71 participants (29 males) were prospectively enrolled, and thirty-five (49.3%) of these individuals were in the group designated as high intima-media thickness (IMT). RESULTS: Multivariate logistic regression analysis revealed three independent risk factors of high IMT: higher levels of anti-HCMV antibody (odds ratio [OR] 1.04, p=0.003), Framingham score (OR 1.14, p=0.018), and levels of IL-1ß (OR 2.96, p=0.013). Anti-HCMV antibody levels had a significantly positive correlation with max-IMT (r=0.523, p<0.001), free T4 levels (r=0.315, p=0.021), and Log(TNF-α) (r=0.562, p<0.001) in multivariate correlation analysis. CONCLUSIONS: These findings may provide insight into the role of HCMV in the pathogenesis of atherosclerosis and chronic inflammation in elderly individuals.

Reassessing the link between herpes zoster ophthalmicus and stroke.

This editorial will assess a proposed link between herpes zoster ophthalmicus and subsequent stoke. Herpes zoster (also called shingles) is caused by varicella-zoster virus (VZV), one of the 9 human herpesviruses. When children contract their primary VZV infection, virus often travels to the trigeminal ganglia and establishes latency. Upon reactivation in late adulthood, the same virus travels anterograde to cause herpes zoster ophthalmicus. In some people, the virus also traffics from the same trigeminal ganglion along afferent fibers around the carotid artery and its branches. Subsequently VZV-induced inflammation within the affected cerebral arteries leads to occlusion and stroke. In one retrospective analysis of people with herpes zoster ophthalmicus, there was a 4.5 fold higher risk of stroke than in a control group. Two other studies found a less compelling association.

Dysfunctional HDL and progression of atherosclerosis in HIV-1-infected and -uninfected adults.

BACKGROUND: HDL function rather than absolute level may be a more accurate indicator for risk of developing atherosclerosis. Dysfunctional HDL has increased redox activity and reduced antioxidant properties, but it is unknown whether abnormal HDL function is associated with progression of atherosclerosis in HIV-1-infected subjects. FINDINGS: We retrospectively measured serum HDL function in 91 subjects from a prospective 3-year study of carotid artery intima-media thickness (CIMT), which enrolled triads of risk factor-matched persons that were HIV-1-uninfected (n=36) or HIV-1+ with (n=29) or without (n=26) protease inhibitor (PI)-based therapy for ≥ 2 years. HDL function was assessed using a biochemical assay that measures the oxidation of dihydrorhodamine 123 (DHR oxidation rate, DOR), in which higher DOR readout corresponds to dysfunctional HDL phenotype.There were no significant associations between DOR and HIV-1 infection. In univariate analysis of 55 HIV-1-infected subjects, greater waist circumference and lower serum HDL were significantly associated with higher baseline levels of DOR (p=0.01). These subjects had significant increases in levels of DOR over time (3 years) that were associated with white race (p=0.03), higher nadir CD4 count (p<0.001), and lower baseline CIMT (p<0.001). Lower baseline HDL levels, but not function of HDL (p>0.1) (DOR), were significantly associated (p=0.02) with progression of CIMT. CONCLUSION: In a small matched cohort study of HIV-1-infected subjects who had a low cardiovascular risk profile, HDL function changed over time and was independently associated with anthropometric parameters of obesity but not with progression of CIMT.

Detection of human herpesvirus 6 DNA but not human herpesvirus 7 or 8 DNA in atherosclerotic and nonatherosclerotic vascular tissues.

INTRODUCTION: Various viral infections are thought to play a role in the development of atherosclerosis. A number of studies suggest that certain viruses from the Herpesviridae family in particular may lead to atherosclerosis. METHODS: We investigated the presence of human herpesvirus 6 (HHV-6), human herpesvirus 7 (HHV-7), and human herpesvirus 8 (HHV-8) DNA in carotid, iliac, and coronary artery specimens obtained from a group of adult autopsy cases by means of polymerase chain reaction (PCR) analysis and nested PCR techniques. A 28-subject study group with at least type IV atherosclerosis and a 25-subject control group with no visible atherosclerosis were enrolled. RESULTS: HHV-6 DNA was found in the carotid artery specimen of 1 subject with atherosclerosis, in an iliac artery specimen of another subject, and in the iliac artery specimen of one of the control subjects. HHV-7 or HHV-8 DNA was not found in either the atherosclerosis or control cases. CONCLUSIONS: This study is the first to demonstrate the presence of HHV-6 in atherosclerotic vascular tissues. HHV-7 and HHV-8 were not found in atherosclerotic tissues; however, further research on broader study groups and with different protocols is needed to determine whether these viruses play a role in the formation of atherosclerosis.

Infectious burden and carotid plaque thickness: the northern Manhattan study.

BACKGROUND AND PURPOSE: The overall burden of prior infections may contribute to atherosclerosis and stroke risk. We hypothesized that serological evidence of common infections would be associated with carotid plaque thickness in a multiethnic cohort. METHODS: Antibody titers to 5 common infectious microorganisms (ie, Chlamydia pneumoniae, Helicobacter pylori, cytomegalovirus, and herpesvirus 1 and 2) were measured among stroke-free community participants and a weighted index of infectious burden was calculated based on Cox models previously derived for the association of each infection with stroke risk. High-resolution carotid duplex Doppler studies were used to assess maximum carotid plaque thickness. Weighted least squares regression was used to measure the association between infectious burden and maximum carotid plaque thickness after adjusting for other risk factors. RESULTS: Serological results for all 5 infectious organisms were available in 861 participants with maximum carotid plaque thickness measurements available (mean age, 67.2+/-9.6 years). Each individual infection was associated with stroke risk after adjusting for other risk factors. The infectious burden index (n=861) had a mean of 1.00+/-0.35 SD and a median of 1.08. Plaque was present in 52% of participants (mean, 0.90+/-1.04 mm). Infectious burden was associated with maximum carotid plaque thickness (adjusted increase in maximum carotid plaque thickness 0.09 mm; 95% CI, 0.03 to 0.15 mm per SD increase of infectious burden). CONCLUSIONS: A quantitative weighted index of infectious burden, derived from the magnitude of association of individual infections with stroke, was associated with carotid plaque thickness in this multiethnic cohort. These results lend support to the notion that past or chronic exposure to common infections, perhaps by exacerbating inflammation, contributes to atherosclerosis. Future studies are needed to confirm this hypothesis and to define optimal measures of infectious burden as a vascular risk factor.

Human immunodeficiency virus per se exerts atherogenic effects.

OBJECTIVE: Premature atherosclerosis in HIV-infected patients has been attributed to highly active antiretroviral therapy (HAART) and the associated metabolic complications. Whether HIV per se plays a role is an unresolved issue. The purpose of this study was to evaluate whether HIV per se exerts atherogenic effects. METHODS: We measured carotid intima-media thickness (IMT) and brachial endothelial-dependent (FMD) and endothelial-independent (NMD) vasodilation in 38 naïve untreated HIV-infected patients and 41 healthy control subjects. RESULTS: Control subjects were selected as to match the HIV patients for metabolic risk factors. Mean carotid IMT was higher in HIV patients (0.85+/-0.2mm; p<0.001) than in controls (0.63+/-0.1mm). In a stepwise multiple regression model, the changes in carotid IMT were predicted by the duration of HIV infection (p<0.001) and CD4 T-cells (p=0.035). Brachial FMD was impaired in HIV patients (8.8+/-3% versus 12.2+/-3% in controls; p<0.001). In contrast, NMD values practically overlapped in the HIV patients and controls. Analysis of the data in relation to viral load showed that FMD was significantly more impaired in the subgroup of patients with viral load values above the median (p<0.001). In addition, there was a highly significant, inverse correlation between FMD and the HIV-RNA copies (p<0.001). CONCLUSION: HIV infection causes functional and structural vascular alterations in a very early stage of the infection independent of HAART and metabolic factors. The data lend support to the viral infectious theory of atherosclerosis. Early assessment of the vascular status in HIV-infected patients is suggested.

Targeting vascular injury using Hantavirus-pseudotyped lentiviral vectors.

Restenosis is a pathological condition involving intimal hyperplasia and negative arterial remodeling. Gene therapy vectors have shown modest therapeutic effects, but the level of infectivity has been relatively poor. In the present study we have designed a modified lentiviral vector (LV) pseudotyped with a strain of Hantavirus (HTNV) to improve the transduction efficiency into vascular smooth muscle and endothelial cells in vitro and in vivo. In vivo studies using adult New Zealand White rabbits demonstrated that local delivery of HTNV-pseudotyped LV (2 x 10(7) TU) into balloon-injured carotid arteries led to highly efficient transduction into endothelial and smooth muscle cells more effectively than VSV-G-pseudotyped LV (2 x 10(7) TU) or replication-defective adenoviral vectors (1-1.5 x 10(9) pfu) as determined by beta-gal immunohistochemistry. Overexpression of extracellular superoxide dismutase in balloon-injured carotid arteries 6 weeks after LV administration resulted in a significant reduction (P = 0.0024) of the intima/media ratio (0.18 +/- 0.09; n = 4) compared to vehicle-infused carotid arteries (0.69 +/- 0.08; n = 7). No beta-gal immunostaining was detected in other systemic organs, including the spleen, liver, heart, lung, kidneys, and brain. Moreover, no changes in plasma alanine aminotransferase or aspartate aminotransferase were detected following LV administration. In all, these data show that LV pseudotyped with Hantaviral glycoproteins can be a useful vector for targeting therapeutic genes to the vasculature in vivo.

Epstein Barr virus specific T-cells generated from unstable human atherosclerotic lesions: Implications for plaque inflammation.

OBJECTIVE: T-cell activation is an essential feature of atherosclerotic plaque inflammation, which eventually may lead to plaque rupture. In this study, we investigated if EBV, a common herpes virus, is capable of stimulating atherosclerotic plaque derived T-cells and thus could contribute to atherosclerotic plaque inflammation. METHODS: Plaque derived T-cell cultures were established from symptomatic carotid atherosclerotic plaques of 19 patients. B-cells from the same patients were transformed with EBV to form lymphoblastoid cell lines (B-LCL) that served as antigen presenting cells. The proliferation of T-cells in the presence of autologous B-LCL was analyzed using 3H-thymidine incorporation. The presence of EBV in atherosclerotic material was analyzed by PCR. RESULTS: Of the 19 cell obtained T-cell cultures, 11 responded to EBV (58%, mean stimulation index: 10.1+/-3.1). PCR analysis showed that EBV DNA was present in 15 of the tissue samples (79%). All the specimens that contained EBV responding T-cells also contained EBV. EBV specific T-cells secreted granzymes, as indication of functional cytotoxic potential. CONCLUSIONS: EBV-specific cytotoxic T-cells and EBV DNA can be frequently observed in human atherosclerotic plaques. This suggests that a T-cell response against EBV could contribute to plaque inflammation, and thus to the onset of acute clinical symptoms.

Detection of cytomegalovirus and Helicobacter pylori DNA in arterial walls with grade III atherosclerosis by PCR.

It has been suggested that some microorganisms may play a role in the etiology or progression of atherosclerotic plaques. The purpose of this study was to assess for the presence of Helicobacter pylori and cytomegalovirus (CMV) DNA using polymerase chain reaction (PCR) technique in vascular-wall specimens obtained during autopsy. Four to 5 mm long samples from 3 different vascular wall specimens (coronary, carotid and abdominal aortas) of 30 patients (23 male, 7 female) were taken for pathologic and microbiologic investigations during autopsy. H. pylori DNA was found in 48.2% atherosclerotic and 19.6% non-atherosclerotic vascular wall specimens, whereas CMV DNA was found in 37.9% atherosclerotic and 32.7% non-atherosclerotic vascular wall specimens. In terms of CMV DNA detection, no statistically significant differences between the atherosclerotic and non-atherosclerotic groups were present (P > 0.05). However, there was a statistically significant difference between the atherosclerosis and non-atherosclerotic groups in terms of H. pylori DNA in coronary and abdominal aorta arteries (p = 0.016 and p = 0.0029 respectively) but not in carotid arteries (p = 1.00). In conclusion, the correlation between H. pylori and atherosclerosis could be suggested. These finding warrant further investigation regarding the role of H. pylori in atherosclerosis.

Detection of herpes simplex virus, cytomegalovirus and Epstein-Barr virus DNA in atherosclerotic plaques and in unaffected bypass grafts.

BACKGROUND: Herpes virus infections are suspected to be involved in the pathogenesis of atherosclerosis. OBJECTIVE AND METHOD: Viral DNA of herpes simplex virus (HSV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) was analyzed by real-time PCR on 48 biopsies from atherosclerotic plaques extracted by end-arterectomy (46 coronary arteries, 2 carotid arteries), and in tissue from non-atherosclerosis vessels from the same patient as controls (23 internal mammary arteries, 43 saphenous veins). RESULTS: HSV-1 DNA was detected significantly more frequently in plaques (35%) than in control veins (9%, P = 0.006). However, the frequency of HSV-1 DNA detection in the internal mammary artery grafts was as high as in plaques (22%, P = 0.28). CMV and EBV DNA were exclusively found in plaques but not in controls, with 10% for CMV (P = 0.06 versus veins, P = 0.17 versus graft arteries) and 2% for EBV (P = 1.0), respectively. HSV-2 was neither detected in plaques nor in controls. Herpes viral DNA was significantly associated only with arterial hypertension but not with other classical risk factors (P = 0.02), in accordance with the hypothesis that herpes viral infection may alter the vessel wall. CONCLUSION: We conclude that herpes viral infections may have a role in atherosclerosis and that the presence of herpes viral DNA in the grafts used for bypass surgery might constitute a potential risk for atherosclerosis or restenosis.

[Expression of human cytomegalovirus immediate early gene in the intracranial artery walls of atherosclerosis].

OBJECTIVE: Human cytomegalovirus (HCMV), especially the immediate early (IE) gene of the virus, has been implicated in the pathogenesis of atherosclerosis. The aim of this study was to confirm the presence of HCMV IE gene DNA in intracranial artery walls and the association of the virus with the development of atherosclerosis. METHODS: HCMV IE gene was tested in formaldehyde-fixed intracranial arteries from 35 cases with cerebral atherosclerosis and 20 negative controls. In situ hybridization as well as polymerase chain reaction (PCR) was used to detect the presence of DNA in sections of paraffin-embedded tissue samples. Probes and primers were derived from major immediate early (MIE) genomic regions of cytomegalovirus strain AD169. RESULTS: The DNA of HCMV was found in 40.0% and 10.0% of arterial walls with atherosclerosis and negative control group by in situ hybridization, respectively, in 60.0% and 30.0% by PCR, respectively. Significant deference was found between them (P=0.018, P=0.032). There was also significant difference between grade III-IV and grade I-II atherosclerosis by both methods (P=0.027, P=0.009). CONCLUSION: The results suggested that HCMV IE DNA exists in the atherosclerotic arterial walls, therefore, there might be an association between the IE gene in intracranial artery walls and the atherosclerosis. The arterial wall with the smooth muscle cells, might be the potential site of the virus persistence. HCMV may play a role in the pathogenesis of the atherosclerosis.

Cell-type-specific characteristics modulate the transduction efficiency of adeno-associated virus type 2 and restrain infection of endothelial cells.

Adeno-associated viruses (AAVs) are promising vectors for various gene therapy applications due to their long-lasting transgene expression and wide spectrum of target cells. Recently, however, it has become apparent that there are considerable differences in the efficiencies of transduction of different cell types by AAVs. Here, we analyzed the efficiencies of transduction and the transport mechanisms of AAV type 2 (AAV-2) in different cell types, emphasizing endothelial cells. Expression analyses in both cultured cells and the rabbit carotid artery assay showed a remarkably low level of endothelial cell transduction in comparison to the highly permissive cell types. The study of the endosomal pathways of AAV-2 with fluorescently labeled virus showed clear targeting of the Golgi area in permissive cell lines, but this phenomenon was absent in the endothelial cell line EAhy-926. On the other hand, the response to the block of endosomal acidification by bafilomycin A1 also showed differences among the permissive cell types. We also analyzed the effect of proteasome inhibitors on endothelial cells, but their impact on the primary cells and in vivo was not significant. On the contrary, analysis of the expression pattern of heparan sulfate proteoglycans (HSPGs), the primary receptors of AAV-2, revealed massive deposits of HSPG in the extracellular matrix of endothelial cells. The matrix-associated receptors may therefore compete for virus binding and reduce transduction in endothelial cells. Accordingly, in endothelial cells detached from their matrix, AAV-2 transduction was significantly increased. Altogether, these results point to a more complex cell-type-specific mode of transduction of AAV-2 than previously appreciated.