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.

The culture of primary duck endothelial cells for the study of avian influenza.

BACKGROUND: Endothelial cells play a major role in highly pathogenic avian influenza (HPAI) virus pathogenesis in gallinaceous poultry species (e.g. chicken, turkey and quail). Upon infection of gallinaceous poultry with HPAI viruses, endothelial cells throughout the body become rapidly infected, leading to systemic dissemination of the virus, disseminated intravascular coagulation, oedema and haemorrhaging. In contrast, the pathogenesis of HPAI viruses in most wild bird species (e.g. duck, goose and gull species) is not associated with endothelial tropism. Indeed, viral antigen is not found in the endothelial cells of most wild bird species following infection with HPAI viruses. This differential endothelial cell tropism in avian species is poorly understood, mainly due to the absence of appropriate cell culture systems. RESULTS: Here, we describe the isolation and purification of primary duck endothelial cells from the aorta or bone marrow of Pekin duck embryos. Cells were differentiated in the presence of vascular endothelial growth factor and, if needed, enriched via fluorescent-activated cell sorting based on the uptake of acetylated low-density lipoprotein. The expression of von Willebrand factor, a key marker of endothelial cells, was confirmed by polymerase chain reaction. Monocultures of duck endothelial cells, either derived from the aorta or the bone marrow, were susceptible to infection with an H5N1 HPAI virus but to a much lesser extent than chicken endothelial cells. CONCLUSIONS: The methods described herein to isolate and purify duck endothelial cells from the aorta or bone marrow could also be applied to obtain microvascular endothelial cells from other tissues and organs, such as the lung or the intestine, and represent a valuable tool to study the pathogenesis of avian viruses.

Murine Norovirus Infection Variably Alters Atherosclerosis in Mice Lacking Apolipoprotein E.

Macrophages play a key role in the development of atherosclerosis. Murine noroviruses (MNV) are highly prevalent in research mouse colonies and infect macrophages and dendritic cells. Our laboratory found that MNV4 infection in mice lacking the LDL receptor alters the development of atherosclerosis, potentially confounding research outcomes. Therefore, we investigated whether MNV4 likewise altered atherosclerosis in ApoE(-/-) mice. In the presence of oxidized LDL, MNV4 infection of ApoE(-/-) bone marrow-derived macrophages increased the gene expression of the inflammatory markers inducible nitric oxide synthase, monocyte chemoattractant protein 1, and IL6. In addition, proteins involved in cholesterol transport were altered in MNV4-infected ApoE -/- bone marrow-derived macrophages and consisted of increased CD36 and decreased ATP-binding cassette transporter A1. MNV4 infection of ApoE(-/-) mice at 12 wk of age (during the development of atherosclerosis) had a variable effect on atherosclerotic lesion size. In one study, MNV4 significantly increased atherosclerotic plaque area whereas in a second study, no effect was observed. Compared with controls, MNV4-infected mice had higher circulating Ly6C-positive monocytes, and viral RNA was detected in the aortas of some mice, suggesting potential mechanisms by which MNV4 alters disease progression. Plaque size did not differ when ApoE -/- mice were infected at 4 wk of age (early during disease development) or in ApoE -/- mice maintained on a high-fat, high-cholesterol diet. Therefore, these data show that MNV4 has the potential to exert a variable and unpredictable effect on atherosclerosis in ApoE(-/-) mice. We therefore propose that performing experiments in MNV-free mouse colonies is warranted.

Extracellular but not cytosolic superoxide dismutase protects against oxidant-mediated endothelial dysfunction.

Superoxide (O2 (•-)) contributes to the development of cardiovascular disease. Generation of O2 (•-) occurs in both the intracellular and extracellular compartments. We hypothesized that the gene transfer of cytosolic superoxide dismutase (SOD1) or extracellular SOD (SOD3) to blood vessels would differentially protect against O2 (•-)-mediated endothelial-dependent dysfunction. Aortic ring segments from New Zealand rabbits were incubated with adenovirus (Ad) containing the gene for Escherichia coli ß-galactosidase, SOD1, or SOD3. Activity assays confirmed functional overexpression of both SOD3 and SOD1 isoforms in aorta 24 h following gene transfer. Histochemical staining for ß-galactosidase showed gene transfer occurred in the endothelium and adventitia. Next, vessels were prepared for measurement of isometric tension in Kreb's buffer containing xanthine. After precontraction with phenylephrine, xanthine oxidase impaired relaxation to the endothelium-dependent dilator acetylcholine (ACh, max relaxation 33±4% with XO vs. 64±3% without XO, p<0.05), whereas relaxation to the endothelium-independent dilator sodium nitroprusside was unaffected. In the presence of XO, maximal relaxation to ACh was improved in vessels incubated with AdSOD3 (55±2%, p<0.05 vs. control) but not AdSOD1 (34±4%). We conclude that adenoviral-mediated gene transfer of SOD3, but not SOD1, protects the aorta from xanthine/XO-mediated endothelial dysfunction. These data provide important insight into the location and enzymatic source of O2 (•-) production in vascular disease.

Differential identification of atypical pneumonia pathogens in aorta and internal mammary artery related to ankle brachial index and walking distance.

BACKGROUND: We studied the existence of agents in aorta biopsies, such as Chlamydia pneumoniae, cytomegalovirus, and Mycoplasma pneumoniae, that are thought to have a role in atherosclerosis etiopathogenesis role, and their association with peripheral artery disease. MATERIALS AND METHODS: We examined aorta wall and internal mammarian artery (IMA) biopsies taken from two different places in 63 patients in whom coronary artery bypass was performed. In these biopsies, we evaluated the deoxyribonuclease (DNA) of these microorganisms using polymerase chain reaction. From the same patients, we recorded the ankle brachial index, road walking distance information, lipid profile, C-reactive proteins, blood parameters such as fibrinogen, and the patient's operation data. RESULTS: In the nine aorta biopsies taken from 63 patients, we isolated C pneumoniae DNA. In IMA biopsies taken from the same patients, we detected no microorganism DNA (P < 0.001). In the same aorta biopsies, we found no cytomegalovirus or M pneumoniae DNA. We examined 12 patients using an index value of 0.9 in the ankle brachial index evaluation; eight had C pneumoniae in the aorta biopsies (P < 0.001). CONCLUSIONS: We found a significant relationship between C pneumoniae DNA and the existence of peripheral artery disease. In the development of atherosclerosis with C pneumoniae, there may be a determinant pathogen in both the aorta and the peripheral arteries. The nonexistence of C pneumoniae DNA in the IMA biopsies may indicate infectious agents because of the predominant endothelial functions in this artery, and thus its resistance to atherosclerosis.

Propagation of classical swine fever virus in vitro circumventing heparan sulfate-adaptation.

Amplification of natural virus isolates in permanent cell lines can result in adaptation, in particular enhanced binding to heparan sulfate (HS)-containing glycosaminoglycans present on most vertebrate cells. This has been reported for several viruses, including the pestivirus classical swine fever virus (CSFV), the causative agent of a highly contagious hemorrhagic disease in pigs. Propagation of CSFV in cell culture is essential in virus diagnostics and research. Adaptation of CSFV to HS-binding has been related to amino acid changes in the viral E(rns) glycoprotein, resulting in viruses with altered replication characteristics in vitro and in vivo. Consequently, a compound blocking the HS-containing structures on cell surfaces was employed to monitor conversion from HS-independency to HS-dependency. It was shown that the porcine PEDSV.15 cell line permitted propagation of CSFV within a limited number of passages without adaptation to HS-binding. The selection of HS-dependent CSFV mutants was also prevented by propagation of the virus in the presence of DSTP 27. The importance of these findings can be seen from the altered ratio of cell-associated to secreted virus upon acquisition of enhanced HS-binding affinity, a phenotype proposed previously to be related to virulence in the natural host.

Nifedipine inhibits vascular smooth muscle cell dedifferentiation via downregulation of Akt signaling.

Calcium is an essential signaling molecule that controls vascular smooth muscle cell (VSMC) contraction, proliferation, and differentiation. Here, we show that the calcium antagonist nifedipine inhibits VSMC dedifferentiation in vitro and in vivo. Differentiated VSMCs cultured on laminin-coated dishes were transferred to laminin-free dishes to induce dedifferentiation. Induction of dedifferentiation resulted in the upregulation of nonmuscle myosin heavy chain expression, a marker of dedifferentiation, and the downregulation of smooth muscle myosin heavy chain expression, a marker of differentiation. Nifedipine significantly inhibited both the induction of these phenotypic changes and upregulation of Akt signaling in these cells. Administration of nifedipine at a low concentration that did not affect blood pressure could inhibit the increase in nonmuscle myosin heavy chain expression and decrease in smooth muscle myosin heavy chain expression in a rat balloon-injury model. Furthermore, nifedipine suppressed neointimal hyperplasia and upregulation of Akt signaling. However, phospho-Akt expression was not suppressed in the regenerating arterial endothelium of the nifedipine-treated rats. The inhibitory effect of the downregulation of Akt signaling by dominant-negative Akt on the induction of VSMC dedifferentiation in the intima was identical to that of nifedipine. In contrast, upregulation of Akt signaling by transfection of the cells with a constitutively active Akt reversed the nifedipine-induced inhibition of VSMC dedifferentiation. In conclusion, nifedipine inhibits VSMC dedifferentiation by suppressing Akt signaling, thereby preventing neointimal thickening.

Classical swine fever virus infection protects aortic endothelial cells from pIpC-mediated apoptosis.

Classical swine fever virus (CSFV) causes severe disease in pigs associated with leukopenia, haemorrhage and fever. We show that CSFV infection protects endothelial cells from apoptosis induced by the dsRNA mimic, pIpC, but not from other apoptotic stimuli, FasL or staurosporine. CSFV infection inhibits pIpC-induced caspase activation, mitochondrial membrane potential loss and cytochrome c release as well as the pro-apoptotic effects of truncated Bid (tBid) overexpression. The CSFV proteins N(pro) and E(rns) both contribute to CSFV inhibition of apoptosis. We conclude that CSFV infection can inhibit apoptotic signalling at multiple levels, including at the caspase-8 and the mitochondrial checkpoints. By supporting viral replication, endothelial cells may promote CSFV pathogenesis.

Vascular oxidative stress and nitric oxide depletion in HIV-1 transgenic rats are reversed by glutathione restoration.

Human immunodeficiency virus (HIV)-infected patients have a higher incidence of oxidative stress, endothelial dysfunction, and cardiovascular disease than uninfected individuals. Recent reports have demonstrated that viral proteins upregulate reactive oxygen species, which may contribute to elevated cardiovascular risk in HIV-1 patients. In this study we employed an HIV-1 transgenic rat model to investigate the physiological effects of viral protein expression on the vasculature. Markers of oxidative stress in wild-type and HIV-1 transgenic rats were measured using electron spin resonance, fluorescence microscopy, and various molecular techniques. Relaxation studies were completed on isolated aortic rings, and mRNA and protein were collected to measure changes in expression of nitric oxide (NO) and superoxide sources. HIV-1 transgenic rats displayed significantly less NO-hemoglobin, serum nitrite, serum S-nitrosothiols, aortic tissue NO, and impaired endothelium-dependent vasorelaxation than wild-type rats. NO reduction was not attributed to differences in endothelial NO synthase (eNOS) protein expression, eNOS-Ser1177 phosphorylation, or tetrahydrobiopterin availability. Aortas from HIV-1 transgenic rats had higher levels of superoxide and 3-nitrotyrosine but did not differ in expression of superoxide-generating sources NADPH oxidase or xanthine oxidase. However, transgenic aortas displayed decreased superoxide dismutase and glutathione. Administering the glutathione precursor procysteine decreased superoxide, restored aortic NO levels and NO-hemoglobin, and improved endothelium-dependent relaxation in HIV-1 transgenic rats. These results show that HIV-1 protein expression decreases NO and causes endothelial dysfunction. Diminished antioxidant capacity increases vascular superoxide levels, which reduce NO bioavailability and promote peroxynitrite generation. Restoring glutathione levels reverses HIV-1 protein-mediated effects on superoxide, NO, and vasorelaxation.

Chronic immune reactivity against persisting microbial antigen in the vasculature exacerbates atherosclerotic lesion formation.

OBJECTIVE: The purpose of this study was to examine the relative contribution of different immunopathological mechanisms during murine cytomegalovirus (MCMV)-mediated acceleration of atheroma formation in apolipoprotein E-deficient (apoE-/-) mice. METHODS AND RESULTS: To distinguish between the effects of systemic activation and cognate immune reactivity against a pathogen-derived persisting antigen in the vasculature, we used hypercholesterolemic transgenic mice constitutively expressing the beta-galactosidase (beta-gal) transgene in the cardiovascular system (apoE-/- x SM-LacZ). After infection with beta-gal-recombinant MCMV-LacZ, apoE-/-, and apoE-/- x SM-LacZ mice mounted comparable cellular immune responses against the virus. Beta-gal-specific CD(+ T cells expanded rapidly and remained detectable for at least 100 days in both mouse strains. However, compared with apoE-/- mice, apoE-/- x SM-LacZ mice developed drastically accelerated atherosclerosis. Moreover, atherosclerotic lesions in MCMV-LacZ-infected apoE-/- x SM-LacZ but not apoE-/- mice were associated with pronounced inflammatory infiltrates. CONCLUSIONS: Taken together, our data indicate that chronic immune reactivity against pathogen-derived antigens persisting in the vasculature significantly exacerbates atherogenesis.

Cytomegalovirus and proliferative signals in the vascular wall of CABG patients.

OBJECTIVE: To further elucidate the mechanism by which cytomegalovirus (CMV) may promote atherosclerosis, we studied the expression pattern of cellular inflammatory and proliferative signals in the aortic wall of CMV(+) and CMV(-) patients undergoing coronary artery bypass grafting (CABG). METHODS: Aortic biopsies and blood samples of 68 CABG patients were investigated for CMV-DNA by PCR and IN SITU hybridisation. Expression of pp65 antigen, adhesion molecules (ICAM-1, VCAM-1, E-selectin), growth factors (PDGF-AA, TGF-beta), and the cellular proliferation factor Ki-67 was studied by immunohistochemistry. Logistic regression was used to test the correlation between the presence of CMV, vascular inflammation, and traditional noninflammatory risk factors for atherosclerosis. RESULTS: CMV-DNA was detected in the aortic tissue of 52 (76%) patients, and was localised predominantly in vascular smooth muscle cells. In CMV(+) patients, the expression of adhesion molecules and growth factors in the aortic endothelium was increased compared with CMV(-) patients. A positive correlation of elevated CRP, the induction of adhesion molecules and growth factors and CMV(+) was found. Female gender, smoking, and hyperlipidaemia were identified as risk factors for CMV(+). CONCLUSIONS: CMV-DNA in smooth muscle cells induces local growth factor expression as well as endothelial activation, both of which can promote the progression of atherosclerosis. Since traditional atherogenic risk factors increase the likelihood of aortic CMV manifestation, we suggest that CMV plays a crucial role in mediating the progression of atherosclerosis.

[Mesenchymal dysplasia of heart valves, cystic medianecrosis of the aorta and herpetic infection].

Histologic and immunohistochemical studies (use of antibodies for viruses of herpes simplex type 1 and 2, desmin, vimentin, SMA as well as polymerase chain reaction to DNA of viruses of herpes simplex) were made on the material of the valves taken from 1326 patients with valvular heart disease, the ascending aorta of 30 patients with aneurysm, valves of 35 deceased patients without cardiovascular pathology. As a result, expression of viruses of herpes simplex type 1 and/or 2 was found in all cases with mesenchymal dysplasia and cystic medianecrosis in endotheliocytes, fibroblasts, smooth muscle cells of valves and aorta. This indicates the role of these viruses in the pathogenesis of these diseases and their common etiology.

[Strain differences related to Ebola virus reproduction in peritoneal macrophages and in aorta explants of guinea pigs]. / Shtammovye razlichiia reproduktsii virusa Ebola v peritoneal'nykh makrofagakh i éksplantatakh aorty morskikh svinok.

Reproduction of the Ebola strains (ES) virus causing lethality in guinea pigs as well as in peritoneal macrophages and aorta explants of animals was investigated in vitro and in vivo; besides, production of interferon-gamma (IFN-gamma) and of tumor necrosis factor-alpha (TNF-alpha) by macrophages and endotheliocytes of guinea pigs was also studied. The interplay "macrophage--ES" by the example of 2 models of susceptibility to ES demonstrates that the ES lethality is not unambiguously related only with a level of virus reproduction in macrophages. The interplay "endotheliocyte--ES" is indicative of that the ES lethality is inversely dependent on a level of production of the IFN-gamma and of TNF-alpha by endotheliocytes. In general, the Eboly fever lethality is not conditioned only by the ability or inability of ES to reproduce in macrophages and endotheliocytes; it also depends on a variety of pathogenetic factors, one of which could be the cytotoxic action of immune complexes shaping in the process of infection progression.

Interferon regulatory factor-1 mediates PPARgamma-induced apoptosis in vascular smooth muscle cells.

OBJECTIVE: Peroxisome proliferator-activated receptor gamma (PPARgamma) possesses general beneficial effects on the cardiovascular system, such as inhibition of vascular lesion formation and atherosclerosis. However, molecular mechanisms for these effects are yet to be fully defined. The aim of this study is to elucidate whether interferon regulatory factor-1 (IRF-1), a transcriptional factor with anti-proliferative and pro-apoptotic properties, mediates PPARgamma-induced apoptosis in vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: Using Northern and Western blot analyses, we documented that PPARgamma ligands, including ciglitazone, troglitazone, and GW7845, significantly increased IRF-1 expression in VSMCs; however, the PPARalpha ligand (Wy14643) and PPARdelta ligand (GW0742) did not affect its expression. PPARgamma-induced IRF-1 expression was abrogated by pretreatment with the PPARgamma antagonist GW9662. In contrast, adenoviral expression of PPARgamma in VSMCs dramatically increased IRF-1 level. Furthermore, PPARgamma activation increased IRF-1 promoter activity but did not affect IRF-1 mRNA stability. Finally, reducing IRF-1 expression by antisense technology attenuated PPARgamma-induced VSMC apoptosis through decreasing cyclin-dependent kinase inhibitor p21(cip1) and caspase-3 activity. CONCLUSIONS: Our data demonstrate that IRF-1 is a novel PPARgamma target gene and mediates PPARgamma-induced VSMC apoptosis.

Viral mediated gene transfer to sprouting blood vessels during angiogenesis.

Several experimental systems have been applied to investigate the development of new blood vessels. Angiogenesis can be followed ex-vivo by culturing explants of rat aorta 'rings' in biomatrix gels. This angiogenesis system was modified for the study of viral vector mediated gene transfer, using adenovirus, vaccinia- and retroviral vectors. Two modifications were introduced to the model in order to facilitate efficient viral mediated gene transfer, (i) placing the aorta ring on top of a thin layer of collagen such that the angiogenic tissue will be accessible to the viral vector; and (ii) infection of the aorta rings prior to embedding them into the collagen matrix. While adenovirus and vaccinia vectors infected efficiently the aorta rings they induced cell death. Subsequent gene transfer experiments were, therefore, carried with retroviral vectors containing vascular endothelial growth factor (VEGF) and the beta-interferon (IFN) genes. Overexpression of VEGF enhanced significantly microvessel sprouting, while overexpression of IFN-beta induced an antiviral effect. The experimental system described in this study can facilitate the application of other viral vectors to the study of genes that may regulate the complex angiogenic process and thereby open new avenues for vascular gene therapy.

Role of isoprenylcysteine carboxyl methyltransferase in tumor necrosis factor-alpha stimulation of expression of vascular cell adhesion molecule-1 in endothelial cells.

We have previously shown that cytokine stimulation of the expression of vascular cell adhesion molecule-1 (VCAM-1), but not that of intercellular adhesion molecule-1 (ICAM-1), is redox sensitive in endothelial cells. Here, we investigated the role of isoprenylcysteine carboxyl methyltransferase (ICMTase), which methylates isoprenylated CAAX (where C indicates cysteine; A, aliphatic amino acids; and X, almost any other amino acid) proteins, including Rac1, a component of superoxide-generating NAD(P)H oxidase, in the expression of VCAM-1. Pretreatment of endothelial cells with N-acetyl-S-farnesyl-L-cysteine (AFC) or N-acetyl-S-geranylgeranyl-L-cysteine (AGGC), specific inhibitors of ICMTase, inhibited the tumor necrosis factor-alpha (TNF-alpha) stimulation of mRNA expression of VCAM-1 but not that of ICAM-1. Endothelial cells expressed constitutively active ICMTase, as suggested by the presence of methylated Rac1 and the methylation of AFC by the cells. TNF-alpha stimulation of the cells significantly increased the methylation of AFC and Rac1 in endothelial cells. That ICMTase was a component of the redox-sensitive signaling pathway was also suggested by the AFC inhibition of the generation of reactive oxygen species by TNF-alpha. Interestingly, the dominant-negative isoform of Rac1 was not selective but inhibited the TNF-alpha stimulation of the mRNA expression of VCAM-1 and ICAM-1. Thus, ICMTase is a critical component of the redox-sensitive VCAM-1-selective signaling pathway, and it appears to activate a discrete inflammatory signaling pathway, at least in part, through the methylation of Rac1.

Enhanced atherogenesis is not an obligatory response to systemic herpesvirus infection in the apoE-deficient mouse: comparison of murine gamma-herpesvirus-68 and herpes simplex virus-1.

Viral and bacterial infectious agents have been implicated in the etiology of atherosclerosis. We have previously shown that a gamma-herpesvirus can accelerate atherosclerosis in the apolipoprotein E-deficient (apoE-/-) mouse. To address whether a virally induced systemic immune response is sufficient to trigger enhanced atheroma formation, we infected apoE-/- mice with murine gamma-herpesvirus-68 (MHV-68) or herpes simplex virus-1 (HSV-1). In this study, we show that both viruses were able to induce a cell-mediated and humoral immune response in the apoE-/- mouse, which was sustained over a period of 24 weeks. Although intranasal or intraperitoneal infection with MHV-68 induced similar levels of virus-specific IgG1 and IgG2a antibodies in the serum of apoE-/- mice, those infected with HSV-1 showed higher anti-HSV-1 IgG2a compared with IgG1 antibody levels. In addition, viral message was not detected in the aortas of HSV-1-infected animals, whereas we have shown previously that MHV-68 mRNA can be detected in the aortas of infected mice as early as 5 days after infection. Compared with control mice, apoE-/- mice infected with MHV-68 showed accelerated atherosclerosis, whereas mice infected with HSV-1 did not. These data indicate that a systemic immune response to any particular infectious agent is insufficient to induce enhanced atherosclerosis in the apoE-/- mouse and point to specific infections or immune mechanisms that might be essential for virally enhanced atherogenesis.

Novel technique of aortic banding followed by gene transfer during hypertrophy and heart failure.

Aortic banding in the rat has become a popular method to induce left ventricular (LV) hypertrophy and heart failure. However, because of often extensive intrathoracic adhesions and inflammatory cell infiltrates resulting from the traditional surgical approach, an uncomplicated second thoracic incision for genetic manipulation is impeded. In this study, we describe a novel surgical technique of aortic banding which avoids opening the sternum and thereby avoids adhesions and surgery-related inflammation. Placing a clip on the ascending aorta using a suprasternal approach in Sprague-Dawley rats created proximal aortic constriction. The present study was initiated to determine whether a replication-deficient adenovirus would enable efficient gene transfer to adult cardiac myocytes undergoing hypertrophy and transitioning to heart failure. Echocardiography performed at week 24 revealed significant concentric hypertrophy and increased fractional shortening followed by LV dilatation with decreased fractional shortening after 27 wk of banding. An adenoviral solution encoding for the reporter green fluorescent protein gene (GFP) was delivered to the heart. Fluorescent microscopy revealed global gene expression throughout hypertrophied and failing hearts. Our studies demonstrate that a novel suprasternal approach can be applied to create an LV hypertrophy model followed by heart failure which also allows investigators to perform genetic manipulations in vivo through gene transfer without the complication of adhesions and surgical trauma-induced inflammation. Furthermore, our approach to delivery of transgenes results in homogenous gene expression in both hypertrophied and failing hearts.

Herpesvirus (HSV-1, EBV and CMV) infections in atherosclerotic compared with non-atherosclerotic aortic tissue.

The viral nucleic acid of herpes simplex virus type 1 (HSV-1), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) was studied by polymerase chain reaction (PCR), Southern blotting and in situ hybridization (ISH) in aortic tissues from 33 autopsies. In 23 cases involving persons who ranged from 23 weeks to 75 years of age at the time of death, the tissue was histologically non-atherosclerotic. Of these 23, aortic tissues tested positive for HSV-1 in 13%, for EBV in 13% and for CMV in 4%. In the other 10 cases involving persons who were 53-75 years old at death, atherosclerotic aortic tissue tested positive for HSV-1 in 80%, for EBV in 80% and for CMV in 40%. Neither double nor triple infections occurred in the non-atherosclerotic group, whereas six of 10 were positive for two viruses, and two of 10 were positive for three viruses in the atherosclerotic group. By in situ hybridization, the viruses were localized in cells morphologically consistent with endothelial cells and smooth muscle cells. We detected HSV-1, EBV and CMV DNA in cells in the upper portion of the non-atherosclerotic aortic wall, whereas viral DNA was detected more extensively in atherosclerotic lesions than in non-atherosclerotic tissue. We also are the first to show the existence of EBV DNA in the human aortic wall. In conclusion, we suggest that the high incidence and kinds of herpesviruses are related to the high incidence of atherosclerosis.