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Background: Cardiac injury is common in patients who are hospitalized with coronavirus disease 2019 (COVID-19) and portends poorer prognosis. However, the mechanism and the type of myocardial damage associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain uncertain., Methods: We conducted a systematic pathological analysis of 40 hearts from hospitalized patients dying of COVID-19 in Bergamo, Italy, to determine the pathological mechanisms of cardiac injury. We divided the hearts according to presence or absence of acute myocyte necrosis and then determined the underlying mechanisms of cardiac injury., Results: Of the 40 hearts examined, 14 (35%) had evidence of myocyte necrosis, predominantly of the left ventricle. Compared with subjects without necrosis, subjects with necrosis tended to be female, have chronic kidney disease, and have shorter symptom onset to admission. The incidence of severe coronary artery disease (ie, >75% cross-sectional narrowing) was not significantly different between those with and without necrosis. Three of 14 (21.4%) subjects with myocyte necrosis showed evidence of acute myocardial infarction, defined as ≥1 cm 2 area of necrosis, whereas 11 of 14 (78.6%) showed evidence of focal (>20 necrotic myocytes with an area of ≥0.05 mm 2 but <1 cm 2 ) myocyte necrosis. Cardiac thrombi were present in 11 of 14 (78.6%) cases with necrosis, with 2 of 14 (14.2%) having epicardial coronary artery thrombi, whereas 9 of 14 (64.3%) had microthrombi in myocardial capillaries, arterioles, and small muscular arteries. We compared cardiac microthrombi from COVID-19-positive autopsy cases to intramyocardial thromboemboli from COVID-19 cases as well as to aspirated thrombi obtained during primary percutaneous coronary intervention from uninfected and COVID-19-infected patients presenting with ST-segment-elevation myocardial infarction. Microthrombi had significantly greater fibrin and terminal complement C5b-9 immunostaining compared with intramyocardial thromboemboli from COVID-19-negative subjects and with aspirated thrombi. There were no significant differences between the constituents of thrombi aspirated from COVID-19-positive and -negative patients with ST-segment-elevation myocardial infarction., Conclusions: The most common pathological cause of myocyte necrosis was microthrombi. Microthrombi were different in composition from intramyocardial thromboemboli from COVID-19-negative subjects and from coronary thrombi retrieved from COVID-19-positive and -negative patients with ST-segment-elevation myocardial infarction. Tailored antithrombotic strategies may be useful to counteract the cardiac effects of COVID-19 infection.

Competing Interests: Declaration of Competing Interest Kirk U. Knowlton, M.D. –None.

Background: This study investigates morphofunctional adaptations of the heart stroma and parenchyma in rats that are chronically infected with Trypanosoma cruzi., Methods: Four-month-old male Wistar rats were randomized into control (n=14) and infected (n=14) groups. Infected animals were inoculated with T. cruzi Y strain. After 9 weeks, the animals were euthanized, and the right atrium (RA) and left ventricle (LV) were removed for biochemical, stereological, and cardiomyocyte mechanical analyses., Results: Infected animals presented cardiomyocyte atrophy and myocardial fibrosis. For these animals, the total volume, length, surface area, and cross-sectional area of cardiomyocytes were significantly reduced, and the total interstitial and collagen volumes were significantly increased in the RA and LV compared to the controls. The total volume and length of blood vessels were significantly increased in the LV, and the total blood vessel surface area was significantly higher in the RA of infected animals. RA and LV cardiomyocytes from infected animals exhibited a significant reduction in cell shortening (43.02% and 24.98%, respectively), prolongation of the time to the peak of contraction (17.09%) and the time to half relaxation (23.68%) compared to non-infected animals. Lipid hydroperoxides, but not mineral concentrations, were significantly increased in the RA and LV from infected animals, showing an inverse correlation with cell shortening., Conclusions: T. cruzi infection induces global structural remodeling of the RA and LV in rats. This remodeling coexists with cardiomyocyte contractility dysfunction, which is possibly related to the abnormal organization of the myocardial stroma and increased cellular lipid peroxidation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Several lines of evidence suggest the involvement of infectious agents in the pathogenesis of atherosclerosis. Furthermore, a correlation between infection-driven inflammatory burden and acute manifestation of coronary artery disease has been hypothesized. The aim of this work was to assess whether human herpesvirus (HHV)-6 and HHV-8, two DNA viruses with a distinct tropism for endothelium and lymphocytes, may be associated with coronary instability. An age- and gender-matched cross-sectional study was undertaken in 70 patients with testing of plasma HHV-6 and HHV-8 DNA load in different cardiovascular clinical settings: 29 patients with acute myocardial infarction, 21 patients with stable coronary artery disease, and 20 patients without coronary and carotid artery atherosclerosis subjected to cardiac valve replacement. In all patients, HHV-6 and HHV-8 plasma DNA was tested by using highly sensitive, calibrated quantitative real-time PCR assays which employ a synthetic DNA calibrator to adjust for DNA extraction and amplification efficiency. HHV-8 viremia was undetectable in all three groups. HHV-6 viremia was detected in a substantial fraction of the samples examined (18.6%) without significant differences among the three groups (ST segment elevation myocardial infarction: 17.2%; stable coronary artery disease: 14.3%; patients without coronary and carotid artery atherosclerosis: 25%). Furthermore, no significant differences in plasma HHV-6 load were observed amongst the three groups of patients. These findings indicate that coronary instability is not associated specifically with active HHV-6 or HHV-8 infection. However, an unusually high rate of active HHV-6 infection was documented among patients without atherosclerosis admitted to hospital with cardiac disease., (Copyright © 2012 Wiley Periodicals, Inc.)

It has been shown that cytomegalovirus (CMV) is present in coronary atherosclerotic plaques, but the clinical relevance of this presence remains to be elucidated. In this study we sought to examine CMV infection in atherosclerosis patients defined by different methods and to identify the clinical significance of CMV replication in the atherosclerotic plaques. The study included 105 consecutive patients who were admitted to our department and underwent coronary artery bypass grafting (CABG) surgical interventions. Coronary atherosclerotic specimens as well as 53 specimens from the mamillary artery of these same patients were analyzed. Enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) methods were used for evaluations. The CMV PCR test result was positive for 28 (26.7%) of patients with coronary artery atherosclerosis. After adjusting for other risk factors, coronary artery disease patients with a history of acute coronary syndrome were more likely to be positive for CMV PCR test (P=0.027; odds ratio: 4.2; 95% CI: 1.18-15.0). They were also more likely to have a positive family history for cardiovascular diseases (CVD). This study confirms previous evidence about the replication of CMV virus in the atherosclerotic plaques of coronary arteries and brings clinical significance to this observation by showing a higher prevalence of acute coronary syndromes in those patients with CMV-infected plaques. Our study also suggests a familial vulnerability to CMV replication in the coronary artery walls.

Objective: The aim of this study was to investigate whether bacterial and viral infectious agents can be demonstrated in atherosclerotic lesions of patients with coronary artery disease (CAD) as well as in stenotic aortic and mitral valves from patients undergoing heart valve replacement., Methods: In this cross-sectional study, the presence of Chlamydophila pneumoniae, Mycoplasma pneumoniae, Cytomegalovirus (CMV), and Epstein-Barr virus (EBV) was investigated by polymerase chain reaction in atherosclerotic and non-atherosclerotic vascular samples taken from patients undergoing coronary artery bypass surgery due to CAD, and from patients undergoing aortic (AVR) and/or mitral valve replacement (MVR) secondary to valvular stenosis. For statistical analyses ANOVA, Chi-square test or Fisher's exact test were used., Results: The presence of C. pneumoniae, M. pneumoniae, and CMV in atherosclerotic versus non-atherosclerotic samples was as follows: 30% vs. 16.7% (p=0.222), 6.7% vs. 3.3% (p=0.554), and 10% vs. 0% (p=0.076), respectively. In valve group, same pathogens were present in AVR and MVR patients as follows: 24.2% vs. 21.4% (p=0.773), 9.1% vs. 7.1% (p=0.758), and 21.2% vs. 11.9% (p=0.275). EBV DNA was not detected in any of vascular specimens, but in one (3%) patient with AVR (p=0.256)., Conclusion: Our results suggest that C. pneumoniae, M. pneumoniae, and CMV are present with similar frequency both in atherosclerotic and non-atherosclerotic vessels. We conclude that although non-atherosclerotic, vascular samples of CAD patients are invaded by infectious agents as like as atherosclerotic vessels. We further conclude that C. pneumoniae, M. pneumoniae, and CMV are present in stenotic aortic and mitral valves and atherosclerotic tissues with similar frequency indicating that atherosclerosis and valvular stenosis might share a common etiology related to infection.

Background: Increasing evidence supports a link between serological evidence of pathogen burden (PB) and the risk for future cardiovascular events. Our study evaluates the intimal presence of 4 pathogens in atheroma, clinically associated with acute coronary syndromes (ACS) and stable angina (SA), and the effect on the expression of intimal C-reactive protein (CRP), tissue factor (TF) and human heat-shock protein 60 (hHSP60)., Methods: Coronary atherectomy specimens retrieved from 60 primary lesions of patients with ACS (n=35) or SA (n=25) were assessed immunohistochemically for the presence of Chlamydia pneumoniae (Cpn), Helicobacter pylori (HP), Cytomegalovirus (CMV) and Epstein–Barr Virus (EBV) and for the expression of CRP, TF, and hHSP60., Results: Analysis revealed eight lesions without, 22 lesions with one, 19 lesions with two, seven lesions with three, and four lesions with four pathogens. Cpn was present in 73%, HP in 31%, CMV in 16%, and EBV in 40%. Mean value of PB in ACS-lesions was significantly increased. Expressions of CRP, TF, and hHSP60 were significantly higher in ACS lesions. The number of infectious pathogens correlated significant with the expressions of CRP, TF, and hHSP60., Conclusions: Our data demonstrate the impact of PB in plaque instability and suggest local proinflammatory, prothrombotic, and proimmunogenic effects., (© 2010 Elsevier Inc. All rights reserved.)

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.

Objective: To explore the distribution of cytomegalovirus (CMV) in vascular tissues and the relationship between virus and atherosclerogenesis after CMV infecting mice., Methods: (1) C57 BL/6J Murine model of CMV infection was established by intraperitoneal injection of CMV lethiferous amount. (2) After 12 weeks of CMV infection, the sera, carotids, aorta, hearts and postcaval veins from the mice were collected under euthanasia. The tissues would be used to DNA extraction, PCR and pathological examination. (3) Interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) in serum were measured with ILELISA., Results: (1) The typical pathologic feature in 2 aorta samples of 6 mice infected by CMV was found and the mice uninfected by CMV did not show any pathologic change. (2) CMV DNA appeared in 6 aorta, 6 postcaval veins, 4 carotids and 4 heart tissues including endocardium, cardiac muscle and coronary artery from the CMV infected mice. CMV DNA was not found in the vascular and heart tissues from 6 mice uninfected by CMV. (3) The ELISA test showed the significant difference (Mann-Witney test of Nonparametric Test, P < 0.05) in serum IL-6 (Median among 25% and 75% percentile: 113.7 pg/ml vs. 49.77 pg/ml) and MCP-1 (Median among 25% and 75% percentile: 128.7 pg/ml vs. 45.36 pg/ml) between CMV infected mice and uninfected mice., Conclusion: Cardiovascular cells are CMV latent reservoir in host body and CMV infection and the cytokines induced by CMV infection probably relate to atherosclerogenesis.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals principally affecting cattle, pigs and sheep. FMD virus (FMDV) uses the alpha(V)beta(1), alpha(V)beta(3), alpha(V)beta(6), and alpha(V)beta(8) integrins as receptors in vitro via a highly conserved arginine-glycine-aspartic acid amino acid sequence motif located within the betaG-betaH loop of VP1. Immunofluorescence and confocal microscopy were used to study the expression of two major FMDV receptors, alpha(V)beta(3) and alpha(V)beta(6), within epithelial tissues from FMDV-infected and uninfected cattle in order to understand the role of these receptors in tissue tropism. Integrin alpha(V)beta(6) was expressed by epithelial cells in tissues that are important sites for FMDV replication (i.e. tongue and coronary band). Integrin alpha(V)beta(3) was detected in epithelium of all tissues examined except tongue. In addition, alpha(V)beta(3) expression was associated with blood vessels in all tissues examined. In infected tissues, alpha(V)beta(6) integrin was distributed on the surface of those epithelial cells also expressing FMDV antigen. Although integrin alpha(V)beta(3) has been shown to be a receptor for FMDV, no expression of alpha(V)beta(3) was associated with FMDV-positive keratinocytes in the tongue. In contrast, podal epithelial cells containing FMDV antigen also expressed alpha(V)beta(3) integrin. Thus, at the cellular level the expression of these two integrins correlates with susceptibility to infection and may contribute substantially to viral tropism in FMD pathogenesis.

Several studies have reported an association between infectious agents and atherosclerosis. Cytomegalovirus (CMV) is the most commonly implicated viral pathogen. However, the role of CMV in atherosclerosis remains obscure. The present study evaluated the presence of CMV DNA in atherosclerotic plaques and normal vessel walls. A total of 40 arterial specimens from coronary plaques and 27 samples from normal vessels were obtained from 26 patients who underwent aortocoronary bypass surgery. The specimens were analyzed by polymerase chain reaction for the presence of the CMV immediate early genomic region. CMV DNA was detected in 9 out of 26 patients (34.6%). Viral DNA was detected in both nonatherosclerotic tissues and atherosclerotic plaques. No statistically significant differences were observed between normal and diseased vessels. Our findings, in accordance with previous studies, do not support a direct causative role of CMV in the development of atherosclerotic plaques.

We performed in vivo phage display in the stroke prone spontaneously hypertensive rat, a cardiovascular disease model, and the normotensive Wistar Kyoto rat to identify cardiac targeting peptides, and then assessed each in the context of viral gene delivery. We identified both common and strain-selective peptides, potentially indicating ubiquitous markers and those found selectively in dysfunctional microvasculature of the heart. We show the utility of the peptide, DDTRHWG, for targeted gene delivery in human cells and rats in vivo when cloned into the fiber protein of subgroup D adenovirus 19p. This study therefore identifies cardiac targeting peptides by in vivo phage display and the potential of a candidate peptide for vector targeting strategies.

Unlabelled: Our aim was to assess the medium-time results after orthotopic heart transplantation (OHT) depending on the serostatus of heart transplant recipients at the time of transplantation., Materials and Methods: We enrolled 120 patients in this retrospective study including 107 males and an overall mean age of 49.1 +/- 8.6 years ischemic heart disease (IHD) was the diagnosis before OHT in 46%. The mean follow-up was 48 months. The first diagnostic coronary angiography was routinely preformed at 1 year after OHT, and then every second year. We evaluated every incidence of a change in the coronary arteries, the presence of significant stenosis (requiring percutaneous coronary intervention), acute myocardial infarction, death, or retransplantation. Using indirect immunofluorescence assays we detected Epstein-Barr virus (EBV) antigenemia by identification of antibodies against early antigens of EBV (EA) of IgM and IgG classes as well as IgG antibodies against nuclear antigen of EBV (EBNA). Since April 2001 all three types of antibodies were routinely investigated on admission to the hospital for OHT., Results: At the time of transplantation, IgM-EA antibodies were detected in 17 (14%) patients, IgG-EA in 35 (29%), and IgG-EBNA in 113 (94%). Survival rate, incidence of cardiac allograft vasculopathy and death, as a function of the serostatus of the heart recipient at the time of transplantation were estimated by Kaplan-Meier curves; the results were compared by log-rank tests. The findings among IgM EA, IgG EA, and IgG EBNA-positive and -negative groups were statistically comparable., Conclusion: Infection with EBV before OHT did not worsen the prognosis over the first 4 years after transplantation.

Several lines of evidence suggest that dietary fat and cholesterol may play a role in the pathogenesis of human immunodeficiency virus (HIV) infection and disease progression. We examined the effect that an atherogenic diet (AD) high in saturated fatty acids and cholesterol has on disease progression and systemic inflammation in the simian immunodeficiency virus (SIV)-infected macaque model of acquired immunodeficiency syndrome. Macaques fed an AD had significantly more rapid disease progression, resulting in an increased risk of SIV-related death compared with that in control macaques (hazard ratio, 5.4 [95% confidence interval, 1.7-17.0]; P<.001). Peak viral load was higher in the AD group compared with control values, but further statistically significant differences were not detected at viral set point. The baseline plasma interleukin-18 level after 6 months of the AD was predictive of disease progression. Our findings may have important implications for HIV-infected individuals, because they suggest that dietary changes and manipulation of lipid metabolism could offer potential benefits by slowing disease progression.

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.

Background: Viruses have been detected in atherosclerotic and non-atherosclerotic vascular tissues and may be involved in the mechanisms of atherogenesis. In the present study, we investigated the role of herpes simplex virus (HSV) in the early and late stages of coronary artery atherosclerosis., Methods and Results: HSV prevalence was investigated in coronary artery samples from 42 autopsy cases, in which death was related to myocardial infarction (MI), and 28 young age autopsy cases without heart disease, who had died from fatal injuries (young victim group), using nested polymerase chain reaction (nPCR) and the highly sensitive in situ hybridization with tyramide signal amplification (ISH-TSA). HSV was detected by nPCR in 18 out of 42 (43%) myocardial infarction cases and in 7 out of 28 (25%) young victim group cases, respectively. Using ISH-TSA, HSV DNA was detected in the coronary arteries of the MI group in 16 out of 42 (38%) of the cases; the hybridization signal was localized in the nuclei of endothelial cells, the nuclei of smooth muscle cells, the macrophages around the atheroma, and in the lymphocytes infiltrating the vascular wall. In the young victim group, HSV DNA was detected by ISH-TSA in 7 out of 28 (25%) autopsy cases; the signal was localized in the endothelial and the intimal spindle cells of the coronary arteries., Conclusion: The findings of this study suggest that HSV seems to play a significant role in the initiation and progression of coronary atherosclerosis, and may open new perspectives in preventing the development of vascular damage via an appropriate antiviral treatment.

Reversible opening of the endothelial barrier remains a major obstacle when hearts are transfected via the coronary system. Our aim was to establish an experimental system permitting the continuous analysis of vascular transfer of virus in the intact heart. Isolated saline-perfused rat hearts were inverted and covered with a latex cap to collect interstitial transudate (IT) on the pericardial surface. Adenovirus (10(9) pfu/ml) was stably labeled with rhodamine fluorescent dye. Analysis of IT and coronary perfusate revealed that under baseline conditions, adenovirus in the IT reached 75% of its vascular concentration within 3 min. The nitric oxide-donors S-nitroso-N-acetyl penicillamine (SNAP) and bradykinin (BK) were the most effective substances to increase total IT volume and adenoviral interstitial concentration. Perfusion with 9% serum markedly reduced IT volume flow and delayed the SNAP/BK effect. Our findings demonstrate that SNAP and BK effectively increased coronary transfer of adenovirus suggesting that the inverted isolated heart is a suitable model to optimize vascular transfer of virus under standardized conditions.

Ebola virus glycoprotein (EGP) has been implicated for the induction of cytotoxicity and injury in vascular cells. On the other hand, EGP has also been suggested to induce massive cell rounding and detachment from the plastic surface by downregulating cell adhesion molecules without causing cytotoxicity. In this study, we have examined the cytotoxic role of EGP in primary endothelial cells by transduction with a replication-deficient recombinant adenovirus expressing EGP (Ad-EGP). Primary human cardiac microvascular endothelial cells (HCMECs) transduced with Ad-EGP displayed loss of cell adhesion from the plastic surface followed by cell death. Transfer of conditioned medium from EGP-transduced HCMEC into naive cells did not induce loss of adhesion or cell death, suggesting that EGP needs to be expressed intracellularly to exert its cytotoxic effect. Subsequent studies suggested that HCMEC death occurred through apoptosis. Results from this study shed light on the EGP-induced anoikis in primary human cardiac endothelial cells, which may have significant pathological consequences.

Cardiovascular complications in the course of human immunodeficiency virus (HIV) infection are multifactorial and may be caused by the virus itself or by the related opportunistic infections and neoplasms. Highly active antiretroviral therapy (HAART) has prolonged many patients' lives, but many cardiac sequelae of HIV are not affected by HAART and continue to develop even with treatment. In addition, HAART itself causes in a high proportion of patients a metabolic syndrome, characterized by lipodystrophy/lipoatrophy, dyslipidemia and insulin resistance that may be associated with an increase in peripheral artery and coronary artery diseases. Careful cardiovascular evaluation in the course of HIV disease can identify cardiac complications early enough to treat. All HIV-infected patients candidate to antiretroviral therapy and patients already under treatment should undergo an assessment that includes the evaluation of the cardiovascular risk with the available guidelines.

A number of infectious agents have been implicated in the development of vascular diseases such as atherosclerosis and posttransplantation arterial restenosis. Cytomegalovirus (CMV) has been reported to cause obliteration of coronary arteries by a progressive vasculopathy that involves proliferation of medial smooth muscle cells (SMC). In this study, we report that CMV enhances the serum-induced proliferation of human coronary SMC through activation of a superoxide-generating NADPH oxidase. Exposure of SMC to CMV for 2 h was associated with an 80% increase in NADPH oxidase. This increase in oxidase activity was associated with a two-fold increase in serum-induced DNA synthesis (5-bromo-2'-deoxyuridine incorporation) and significant interleukin-8 (IL-8) production by SMC. Diphenylene iodonium, an inhibitor of NADPH oxidase, significantly inhibited CMV-induced IL-8 production and promotion of serum-induced DNA synthesis. Similar effects were seen following pretreatment of SMC with N-acetyl cysteine, a potent antioxidant, suggesting that oxidative stress following CMV exposure might be responsible for triggering the proliferation of SMC. From this study, we conclude that CMV-mediated promotion of SMC growth is redox sensitive and may be mediated by NADPH oxidase., (Copyright 2003 National Science Council, ROC and S. Karger AG, Basel)

We report the case of a 26-year-old man who died suddenly 9 days after an episode of flu. Microscopic examination of the left anterior descending coronary artery showed an eccentric fibroatheromatic plaque complicated by thrombosis, endothelial erosion and extensive T-cell and macrophage infiltration. Frozen sections of the thrombotic coronary segment, analysed for different infective agents by polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR, showed positive amplification for an enteroviral genome. Enteroviral infection may play an important role in coronary plaque instability and may precipitate thrombotic occlusion.

We report the case of a 34-year-old female patient who died 4 days after hospital admission of acute heart failure clinically mimicking ischemic heart disease. Microscopic examination of the heart showed severe myocarditis. Polymerase chain reaction (PCR), including quantitative real-time PCR, disclosed exclusively parvovirus B19 (PVB19), with a high viral load of 4.3x10(5) PVB19 viral genome equivalents per microg myocardial nucleic acid. Radioactive in situ hybridization detected viral genomes in endothelial cells (ECs) predominantly in the venular compartment and (to a lesser degree) in small arteries and arterioles of the heart, but not in cardiac myocytes or other tissue components. Concomitant with EC infection, marked expression of the adhesion molecule E-selectin was noted, accompanied by margination, adherence, penetration, and perivascular infiltration of T lymphocytes. We speculate that, due to the high viral load in cardiac ECs, PVB19 infection of endothelial cells was sufficient to induce impaired coronary microcirculation with secondary cardiac myocyte necrosis., (Copyright 2003, Elsevier Science (USA). All rights reserved.)

It was established that viral particles, like low-density lipoproteins (LDLP), when subjected to some modification changes, lost their ability to be internalized by tissue somatic cells and acquired tropism to macrophage cells. The data, obtained by us by using the polymerase chain reaction (PCR) method, made it possible to assert that atherosclerotic plaques, isolated from vessels of patients with ischemic heart disease (IHD) who underwent coronary bypass, contained RNA of the A(HINI) and AH3N3) influenza viruses. Whereas, the vessel portions, undamaged by atherosclerosis, did not contain any genetic substances of influenza viruses. It was for the first time that an experimentally supported understanding was expressed on that the atherosclerotic plaques serve as a "reservoir" for influenza viruses. It is also suggested that the mentioned plaques can be the carriers of influenza viruses for a long time, thus, prolonging the persistent form of influenza infection in the human body.

Background: Local gene therapy has enormous potential for the treatment of vascular disease. We determined whether diagnostic ultrasound-mediated destruction of plasmid-loaded albumin microbubbles is a feasible and efficient technique for local vascular gene delivery. For gene transfer, we used a phosphomimetic, active endothelial nitric oxide synthase (eNOS) construct in which Ser1177 was replaced by aspartic acid (S1177D) and exhibits a 2-fold higher basal activity than the wild-type enzyme., Methods and Results: Gas-filled microbubbles (3.0 +/- 1.2 microm) were created by sonication of 5% human albumin in the presence of plasmid DNA encoding for LacZ or eNOS S1177D. Porcine coronary arteries were perfused with DNA-loaded albumin microbubbles in vitro, exposed to diagnostic ultrasound (5 seconds), and incubated for a further 24 hours. Detection of the beta-galactosidase in LacZ-transfected vessels revealed a predominant staining of endothelial cells without any functional impairment of vasoreactivity. Western blotting demonstrated the expression of the eNOS S1177D construct in extracts from the transfected segments. Vascular responsiveness was tested with prostaglandin F(2alpha) and the NOS inhibitor N(omega)nitro-L-arginine. Compared with segments treated with the expression plasmid alone, the contractile response to prostaglandin F(2alpha) was impaired in segments transfected with eNOS S1177D, whereas the contractile response to the administration of N(omega)nitro-L-arginine was markedly enhanced., Conclusions: Ultrasound-mediated destruction of eNOS S1177D DNA-loaded albumin microbubbles is a feasible and efficient method for vascular gene transfection. Transfection resulted in significant protein expression and enhanced NO-mediated relaxation of bradykinin-stimulated porcine coronary arteries.

An efficient gene delivery system is a prerequisite for myocardial gene therapy. Among the various procedures studied so far, catheter-based percutaneous gene delivery to the myocardium through the coronary vessels seems the most relevant to routine clinical practice; however, the optimal conditions remain to be determined. We selectively infused adenoviral vectors encoding luciferase (1 x 10(9) PFU) or beta-galactosidase (1 x 10(10) PFU) into coronary arteries of adult rabbits in various experimental conditions. Coronary artery occlusion for 30 sec, during and after adenovirus delivery, was required to observe luciferase activity in the target area of the circumflex artery (4.0 +/- 1.0 x 10(5) vs. 1.1 +/- 0.2 x 10(4) RLU/mg with and without coronary occlusion, respectively, p < 0.01, and 1.0 +/- 0.1 x 10(3) RLU/mg using nonselective infusion). When adenoviruses were delivered using high-pressure infusion (82 +/- 12 vs. 415 +/- 25 mmHg before and during infusion, respectively, p < 0.01), luciferase activity increased to 8.5 +/- 2.5 x 10(5) RLU/mg (p < 0.05 vs coronary occlusion alone). Coronary venous sinus occlusion with saline buffer retroinfusion starting before and during anterograde adenovirus delivery resulted in a further 4.7-fold increase in luciferase activity (4.4 +/- 0.8 x 10(6) RLU/mg, p < 0.01) with 5-25% blue-stained myocytes in the target area, compared with 0-5% with the other procedures. Histamine or VEGF-A(165) pretreatment, used to increase vascular permeability, slightly increased gene transfer efficiency (8.5 +/- 2.0 x 10(5) and 9.0 +/- 2.5 x 10(5) RLU/mg respectively, p < 0.05 vs. coronary occlusion alone). We conclude that catheter-mediated adenoviral gene transfer to cardiac myocytes through coronary vessels can be a very efficient procedure for myocardial gene therapy, particularly when the vector residence time and perfusion pressure in the vessels are increased.

To identify improved adenovirus vectors for cardiovascular gene therapy, a library of adenovirus vectors based on adenovirus serotype 5 (Ad5) but carrying fiber molecules of other human serotypes, was generated. This library was tested for efficiency of infection of human primary vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Based on luciferase, LacZ, or green fluorescent protein (GFP) marker gene expression, several fiber chimeric vectors were identified that displayed improved infection of these cell types. One of the viruses that performed particularly well is an Ad5 carrying the fiber of Ad16 (Ad5.Fib16), a subgroup B virus. This virus showed, on average, 8- and 64-fold-increased luciferase activities on umbilical vein ECs and SMCs, respectively, compared to the parent vector. GFP and lacZ markers showed that approximately 3-fold (ECs) and 10-fold (SMCs) more cells were transduced. Experiments performed with both cultured SMCs and organ cultures derived from different vascular origins (saphenous vein, iliac artery, left interior mammary artery, and aorta) and from different species demonstrated that Ad5.Fib16 consistently displays improved infection in primates (humans and rhesus monkeys). SMCs of the same vessels of rodents and pigs were less infectable with Ad5.Fib16 than with Ad5. This suggests that either the receptor for human Ad16 is not conserved between different species or that differences in the expression levels of the putative receptor exist. In conclusion, our results show that an Ad5-based virus carrying the fiber of Ad16 is a potent vector for the transduction of primate cardiovascular cells and tissues.

Background and Aim: Experimental and clinical data support an infectious cause of atherosclerosis and thereby coronary artery disease. This study was intended to assess the prevalence and possible clinical associations of the presence of cytomegalovirus DNA within coronary samples from patients undergoing coronary artery bypass grafting., Patients and Methods: A coronary thrombendatherectomy was performed in 53 patients with advanced coronary artery disease. Two samples of each atheroma were used for further analysis and pathogen detection., Result: In 30% of patients with advanced coronary artery disease cytomegalovirus DNA was detected in coronary samples as assessed by highly sensitive PCR methods. The occurrence of the virus within the vessels was characterized by an inhomogeneous distribution pattern., Conclusion: Due to an increased proportion of restenotic lesions and a higher degree of calcification in cytomegalovirus-positive lesions, a causative association between the virus presence and mechanisms of restenosis post angioplasty is further supported. Antiviral pharmacological interventions to prevent restenosis in high-risk patients, however, seem not to be justified by the data currently available.

Background: The pathogenesis of HIV-1-related cardiomyopathy is poorly understood, but HIV-1 has been detected in cardiomyocytes. Whether HIV-1 penetrates into the myocardium by infection of coronary artery endothelial cells (CAEC) or using transcellular or paracellular routes across CAEC has not been resolved., Materials and Methods: A model of the CAEC barrier was constructed with primary CAEC (derived from human coronary vessels). Polymerase chain reaction (PCR) assay, infectious assay, and immunofluorescence were employed to show abortive nature of HIV-1 infection of CAEC. Tight junction (TJ) and cell adhesion proteins were visualized by immunofluorescence. The time course of HIV-1 invasion was measured by HIV-1 RNA assay. Inulin permeability assay determined paracellular leakage. Transmission electron microscopy demonstrated virus-induced endothelial vacuolization., Results: Despite a strong display on CAEC of CXCR4 and a lesser expression of CCR3 and CCR5, HIV-1 did not productively replicate in CAEC, as shown by infectious assay, immunofluorescence, and electron microscopy. HIV-1 infection of CAEC was abortive with minimal reverse transcription of strong stop DNA and pol but not full-length or two LTR DNA circles. Upon infection of the model with 1 million RNA copies of HIV-1JR-FL, virus penetration 2 hr postinfection (PI) was negligible but increased by 1,750% 24 hr PI. The paracellular permeability increased during this period by only 25%. Neither AOP-RANTES nor v-MIPII significantly reduced HIV-1JR-FL invasion. Virus infection did not alter the integral TJ protein occludin and the TJ-associated protein ZO-1. HIV-1 exposed CAEC and brain microvascular endothelial cells (BMVEC) developed extensive cytoplasmic vacuolization with retroviral-like particles in the vacuoles., Conclusions: The endothelium is not an impenetrable barrier to HIV-1. The virus opens a transcellular route across coronary and brain endothelia in cytoplasmic vacuoles.

We investigated a possible correlation between the serologic status concerning Cytomegalovirus (CMV), Chlamydia pneumoniae (CP) and Helicobacter pylori (HP) and the occurrence of restenosis in patients undergoing percutaneous transluminal coronary angioplasty for symptomatic coronary artery disease. Tests for anti-CMV IgG, anti-Chlamydia pneumoniae IgG and IgA and HP IgG and IgA were performed with an enzyme-linked immunosorbent assay (ELISA). Restenosis was defined as >/=50% stenosis at follow-up angiography in a vessel with less than 50% stenosis immediately after PTCA. Of 148 patients, 112 (75.7%) were seropositive for CMV, 75 (50.7%) were seropositive for CP and 78 (52.7%) were seropositive for HP. Restenosis occured in 31.8% of patients. CMV seropositivity was established in 74.5% of patients with restenosis versus 76.2% without restenosis (P=0.82), CP seropositivity was established in 44. 7% of patients with restenosis versus 53.5% without restenosis (P=0. 32), HP seropositivity was established in 53.2% of patients with restenosis versus 52.5% without restenosis (P=0.94). In contrast to some earlier studies CMV or HP seropositivity could not be found to be associated with the risk of restenosis after coronary intervention. An association between the serological status of CP and restenosis could also not be established.

Accelerated graft coronary atherosclerosis is the main obstacle to long-term survival in patients who have had a heart transplant. A possible involvement of the human cytomegalovirus (HCMV) in this type of coronary atherosclerosis has been postulated by many authors but has not been definitively demonstrated. In an attempt to clarify the role of HCMV infection in the pathogenesis of this complication, we looked for in situ antigens or DNA of HCMV in 30 coronary artery segments obtained at necropsy from patients who had undergone orthotopic cardiac transplantation at the São Paulo Heart Institute. We tried to correlate these HCMV markers with the presence of inflammation and/or atherosclerosis in histologic sections. The patients were grouped as follows: GI, less than 170 days of graft survival and absent/mild atherosclerosis; GII, more than 170 days of graft survival and absent/mild atherosclerosis; GIII, more than 170 days of graft survival and severe/moderate atherosclerosis (170 days was the shortest graft survival time associated with atherosclerosis). The search for HCMV genome and antigens in the coronary artery sections was performed using immunohistochemistry, in situ hybridization, and polymerase chain reaction in situ techniques. Immunohistochemistry and in situ hybridization revealed no evidence of HCMV in all 30 cases. Polymerase chain reaction in situ revealed scarce HCMV-positive lymphocytes in two cases (one each from GI and GIII) located in the adventitial layer. These findings preclude a direct role for the HCMV in the pathogenesis of accelerated graft coronary atherosclerosis. However, the possibility of an indirect effect of the virus, such as an immune-mediated inflammatory response by the host that increases the expression of histocompatibility antigens, leading to tissue injury, cannot be excluded.

Background: The biological properties of some herpesviruses such as the ability of latent persistency in the host cells and the presence of viral DNA in atherosclerotic lesions, suggest the possible role of herpesviruses in the development of atherosclerosis. Although many authors proved the presence of viral DNA in arterial wall tissue, the role of herpesviruses in the origin and progress of atherogenesis still remains unclear., Objectives: The aim of this study was to confirm the presence of viral DNA in arterial wall and to associate the presence of these viruses with the development of atherosclerosis in patients with ischemic heart disease (IHD)., Study Design: A possible role of HCMV, EBV and HHV6 in the development of atherosclerosis was tested in 244 IHD patients and 87 coronarographically negative controls. The presence of viral DNA in aortic and venous walls, as well as in a peripheral blood samples was tested by the use of polymerase chain reaction (PCR) accompanied by, immunological tests for anti-virus antibodies IgM and IgG types for all experimental groups., Results: The genomic DNA of HCMV was found in 76 and 59%, DNA of EBV in 59 and 50%, and DNA of HHV6 in 0.08 and 0.0%, of arterial walls of IHD patients and non-ischemic control group, respectively. No viral DNA was found in venous samples. Significant association (P < 0.01) has been proved between CMV infection and IHD., Conclusions: Our results suggest that HCMV and EBV can be found in the arterial wall, so that the arterial wall could be a potential site of persistency of those viruses. We also proved a significant association between the presence of HCMV DNA in aortic walls and atherosclerosis. Despite of the high genetic and biological similarity between CMV and HHV6 no substantial role of HHV6 in atherosclerosis has been proved.

Background: Chemotherapeutic treatment for patients with symptomatic coronary artery disease to reduce cardiovascular events may be initiated in response to elevated antibody titers against Chlamydia pneumoniae or cytomegalovirus. How antibody titers are associated with the endovascular presence of these microorganisms is still unclear., Methods and Results: Antibody titers against C pneumoniae (microimmunofluorescence) and cytomegalovirus (ELISA) in patients undergoing primary (coronary desobliterates, n=80) or repeated CABG (occluded vein grafts, n=45) were correlated with the endovascular presence of the 2 microorganisms. C pneumoniae was detected by means of a nested polymerase chain reaction (PCR) and by culturing. Both conventional PCR and quantitative PCR were applied for detection of cytomegalovirus. C pneumoniae (PCR/culture) was detected in 19/9% (15/80 and 7/80) of coronary desobliterates and in 18/11% (8/45 and 5/45) of occluded vein grafts. There was no statistical evidence that IgG values differed between patients with or without C pneumoniae detection who were undergoing primary CABG. In contrast, repeated-CABG patients with a positive PCR (P=0.0027) or C pneumoniae culture (P=0.0018) had distinctly elevated IgG titers compared with patients in whom C pneumoniae was not detected. Cytomegalovirus could not be detected in the examined specimens., Conclusions: Cytomegalovirus infection does not seem to be associated with advanced coronary artery lesions. C pneumoniae antibody titers are not associated with the endovascular presence of C pneumoniae in patients with coronary artery disease. The observed strong association between elevated IgG titers and the detection of C pneumoniae in occluded vein grafts warrants further investigation.

There is a wealth of clinical and experimental evidence indicating the interaction of cytomegalovirus (CMV) infection and rejection in cardiac and other solid organ allografts. A plausible explanation for this association comes from data showing that therapy with biologicals, sepsis, and rejection, all lead to the release of TNF-alpha which, upon binding to its receptor, activates NF-kB. TNF-alpha is also able to stimulate the activity of the CMV-IE enhancer/promoter region. CMV infection of several cell lines leads to NF-kB activation. NF-kB binding sites are present in regulatory regions of various cellular and viral genes, including the IE enhancer region of CMV. In a reciprocal situation, CMV infection, most likely via gamma-interferon, leads to upregulation of MHC antigens in the transplant and, thereby, to increased transplant immunogenicity. Thus, a vicious circle is induced. We have investigated in detail the pathobiology of CMV and allograft vasculopathy (chronic rejection) in experimental animals, using aortic and cardiac allografts as well as a trachea model. The results may be summarized as follows: Infection of the recipient with rat CMV results in an early inflammatory response in the aortic and cardiac allograft vascular adventitia and intima (endothelialitis) and in the airway wall of tracheal allografts. This early inflammatory response leads to enhanced intimal thickness in aortic and cardiac allografts and enhanced luminal occlusion of tracheal allografts. Timewise, this coincides with early activation of intragraft inflammatory leukocytes and increased mRNA of various growth factors and cytokines. When the recipients receive gancyclovir, the enhanced intimal response in aortic and cardiac allografts and luminal occlusion in tracheal allografts is entirely abolished. Gancyclovir treatment dramatically reduces the inflammatory response in the allograft, and thereby growth factor synthesis in response to injury. However, gancyclovir does not prevent the expression of IE antigen of CMV, suggested to inactivate tumor suppressor protein p53 predisposing smooth muscle cells to increased growth. Taken together, the effect of CMV infection on cardiac allograft dysfunction is bidirectional and biphasic. The bidirectional nature emerges from the observations that acute CMV infection may accelerate acute rejection, and, on the other hand, acute alloimmune response-associated cytokine response may activate latent CMV infection. The biphasic effect of CMV on allograft dysfunction refers to its early and late detrimental effects, i.e. during the time of acute and chronic rejection. These two effects of CMV on allograft dysfunction emphasize the need for precise diagnosis of CMV infection in transplant recipients and pre-emptive or prophylactic anti-viral therapy. The benefits of this strategy may not be evident during the early post-transplant period, but 5-10 years after transplantation they manifest as better graft survival.

Background: Restenotic and atherosclerotic lesions often contain smooth muscle cells (SMCs), which display high rates of proliferation and apoptosis. Human cytomegalovirus (HCMV) may increase the incidence of restenosis and predispose to atherosclerosis. Although the mechanisms contributing to these processes are unclear, studies demonstrate that one of the immediate-early (IE) gene products of HCMV, IE2-84, binds to and inhibits p53 transcriptional activity. Given the role of p53 in mediating apoptosis, we studied the ability of IE2-84 to inhibit p53-dependent apoptosis in human coronary artery SMCs., Methods and Results: Apoptosis of SMCs was induced either by use of an adenovirus vector encoding human wild-type p53 protein or by treatment with doxorubicin. HCMV IE1-72 and IE2-84, the major IE proteins of HCMV, were overexpressed separately with adenovirus vectors encoding each protein, and the effects on p53-induced apoptosis were examined by both nick end-labeling (TUNEL) assay and flow cytometry. Expression of IE2-84, but not IE1-72, protected SMCs from p53-mediated apoptosis., Conclusions: These data indicate that an HCMV IE protein antagonizes p53-mediated apoptosis, suggesting a pathway by which HCMV infection predisposes to SMC accumulation and thereby contributes to restenosis and atherosclerosis.

We and others have observational evidence that human cytomegalovirus (HCMV) may be a pathogen in human atherosclerosis and restenosis. We have experimental evidence that HCMV infects human coronary smooth muscle cells and initiates viral replication. Vascular cells generate reactive oxygen species in response to stress (such as infection or reperfusion) and this leads to increased transcription of atherosclerosis-related cellular and viral genes, and to reactivation of latent HCMV. Finally, we found that aspirin can attenuate this augmented gene transcription via direct and indirect effects.

Atherosclerotic cardiovascular disease is generally accepted to be the result of metabolic disturbances. However, recent studies have suggested an infectious agent, especially Chlamydia pneumoniae or cytomegalovirus, to be involved in the pathogenesis of atherosclerosis. Atherosclerotic plaque specimens obtained from patients with coronary disease either by balloon dilatation catheter (13 cases) or atherectomy (16 patients) were examined for the presence of C. pneumoniae and cytomegalovirus. Using two primer pairs for C. pneumoniae, two primer pairs for the identification of unknown bacteria and primer pairs for the detection of immediate early gene E2 and the late genomic region of cytomegalovirus, we were unable to detect the suspected agents. The absence of C. pneumoniae, other bacteria and CMV in coronary atheromas is against the hypothesis of a pathogenetic role of these agents in coronary atheroma formation in the patients studied.

Human cytomegalovirus (CMV) infection of smooth muscle cells generates reactive oxygen species (ROS) and thereby activates nuclear factor kappaB (NFkappaB), which causes expression of viral and cellular genes involved in immune and inflammatory responses. These changes could account for the mounting evidence suggesting that CMV may contribute causally to restenosis and atherosclerosis. We found that CMV induces ROS, at least partly, through a cyclooxygenase-2 (COX-2)-dependent pathway. Moreover, the viral immediate-early (IE) gene products, IE72 and IE84, have the capacity to transactivate the COX-2 promoter. Aspirin and indomethacin, both cyclooxygenase inhibitors as well as direct ROS scavengers, reduce CMV-induced ROS, probably through both of these activities. Sodium salicylate also has antiviral effects as the result of its potent antioxidant properties. Furthermore, by reducing ROS, aspirin and sodium salicylate inhibit CMV-induced NFkappaB activation, the ability of IE72 to transactivate its promoter, CMV IE gene expression after infection of SMCs, and CMV replication in SMCs. This is the first time aspirin has been shown to have antiviral effects. Thus, it is possible that aspirin has previously unrecognized therapeutic effects in various clinical situations, such as in viral infections (when used as an antipyretic agent) and in atherosclerosis (when used as an antiplatelet agent).

The study of development has been revolutionized by the application of molecular techniques, which make it possible to identify factors involved in the developmental process. However, in order to correctly assess the contribution of these growth factors, transcription factors, receptors or signaling molecules, it is necessary to study them in the animal as a whole; it is not enough to conclude that they must be important based on their expression patterns.

We report on the occurrence of parvovirus particles and VP1 (84 kDa) and VP2 (58 kDa) viral antigens in the heart of a case of fatal myocarditis in a fetus of a 26 year old women. Numerous cells containing intranuclear inclusions were identified within the blood vessels of the heart in a close apposition to muscle fibers. These cells were characterized by plentiful mitochondria and were consistent with erythroblasts. Typically, inclusions consisted of electrondense marginated chromatin and granular and amorphous "cores". At higher magnification, parvovirus particles, approximately 23 nm in diameter, were visualized either as relatively small clusters or forming large paracrystalline arrays. Virus buds were never observed. In addition, unusual membrane proliferation was seen. These findings support a notion that parvovirus may invade the fetal heart.

Objective: To examine histologically biopsies from the coronary arteries of patients undergoing coronary artery bypass grafting (CABG) for evidence of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) antigen and to correlate the incidence with pathological and clinical data., Design: Sequential patients undergoing CABG in whom adequate tissue could be obtained for histology., Setting: University teaching hospital., Patients: Forty-six patients were enrolled. Thirty-one provided sufficient tissue and clinical information for the analysis., Methods: Biopsy material was collected in the operating room and prepared immediately for histology and electron microscopy. Slides were prepared by staining with hematoxylin and eosin, Masson trichrome, avidin biotin complex immunoperoxidase for HSV-1 and HSV-2 protein and specific DNA probes for HSV-1 and HSV-2 by hybridization. Clinical data were obtained in structured interviews., Results: Sixty-one per cent of biopsies demonstrated evidence of inflammation, 45% were positive for antigen to HSV-2 and only one to HSV-1. Significant positive correlations were detected between inflammatory cells in the biopsy and a recent history of cold sores and between the presence of the infiltrate and positivity to HSV-2 antigen., Conclusion: A correlation exists between HSV-2 infection and the inflammatory response associated with atherosclerosis.