HCMV-infection in a human arterial organ culture model: effects on cell proliferation and neointimal hyperplasia.

BACKGROUND: The impact of infections with the human cytomegalovirus (HCMV) for the development of atherosclerosis and restenosis is still unclear. Both a clear correlation and no correlation at all have been reported in clinical, mostly serological studies. In our study we employed a human non-injury ex vivo organ culture model to investigate the effect of an in vitro permissive HCMV-infection on cell proliferation and neointimal hyperplasia for a period of 56 days. RESULTS: During routine-nephrectomies parts of renal arteries from 71 patients were obtained and prepared as human organ cultures. Cell free HCMV infection was performed with the fibroblast adapted HCMV strain AD169, the endotheliotropic strain TB40E, and a clinical isolate (AN 365). After 3, 7, 14, 21, 28, 35, and 56 days in culture staining of HCMV-antigens was carried out and reactive cell proliferation and neointimal thickening were analysed. Successful HCMV-infection was accomplished with all three virus strains studied. During the first 21 days in organ culture no cell proliferation or neointimal hyperplasia was detected. At day 35 and day 56 moderate cell proliferation and neointimal hyperplasia was found both in HCMV-infected segments and mock infected controls. Neointimal hyperplasia in productively HCMV-infected segments was lower than in non infected at day 35 and day 56, but relatively higher after infection with the endotheliotropic TB40E in comparison with the two other strains. CONCLUSION: The data do not support the hypothesis that HCMV-infection triggers restenosis via a stimulatory effect on cell proliferation and neointimal hyperplasia in comparison to non infected controls. Interestingly however, even after lytic infection, a virus strain specific difference was observed.

High prevalence of herpes simplex virus DNA in temporal arteritis biopsy specimens.

Giant cell arteritis (GCA) affecting the cranial arteries is a disease of unknown cause that causes blindness, stroke, and other morbidity. Its sudden onset and segmental distribution are suggestive of diseases that involve viral reactivation, and cranial arteries are known to be innervated by ganglia that harbor herpes simplex virus (HSV). We used a high-sensitivity polymerase chain reaction assay to test for HSV DNA in specimens from 39 consecutive temporal artery biopsies performed for suspected GCA. HSV DNA was detected in 21 (88%) of 24 histologically positive and 8 (53%) of 15 histologically negative specimens (P = .027; Fisher exact test). Analysis of 10 renal artery samples from age-matched control subjects using the same assay showed no detectable HSV DNA. We conclude that detectable HSV DNA is correlated with histologically confirmed GCA in this patient population.

Human cytomegalovirus infection in human renal arteries in vitro.

Studies with animal cytomegaloviruses, epidemiological data from humans as well as in vitro studies suggest the involvement of the human cytomegalovirus (HCMV) in the development of atherosclerosis. Cell culture systems are insufficient for examination of the entire pathogenetic process and a satisfactory animal model for HCMV is not available. An organ culture model was established for HCMV infection of human renal arteries in vitro. After infection with three representative HCMV strains, infectious virus was recovered from supernatants until 144 days post-infection with a peak around day 30 due to a long-lasting productive HCMV infection in still vital cells. Differences in cell tropism and kinetics of infection were identified between the HCMV strains. Specifically, differences in infecting endothelial cells and virus penetration into the lamina media were observed. In infected artery segments, but also in some non-infected arteries from seropositive donors, HCMV DNA could be localized by in situ PCR. Nevertheless, HCMV early antigen was detected by immunohistochemistry exclusively in artery segments infected in vitro. The new organ culture model will permit the study of functional and molecular consequences of HCMV infection in a more physiological micro-environment.

Equine endothelial cells support productive infection of equine infectious anemia virus.

Previous cell infectivity studies have demonstrated that the lentivirus equine infectious anemia virus (EIAV) infects tissue macrophages in vivo and in vitro. In addition, some strains of EIAV replicate to high titer in vitro in equine fibroblasts and fibroblast cell lines. Here we report a new cell type, macrovascular endothelial cells, that is infectible with EIAV. We tested the ability of EIAV to infect purified endothelial cells isolated from equine umbilical cords and renal arteries. Infectivity was detected by cell supernatant reverse transcriptase positivity, EIAV antigen positivity within individual cells, and the detection of viral RNA by in situ hybridization. Virus could rapidly spread through the endothelial cultures, and the supernatants of infected cultures contained high titers of infectious virus. There was no demonstrable cell killing in infected cultures. Three of four strains of EIAV that were tested replicated in these cultures, including MA-1, a fibroblast-tropic strain, Th.1, a macrophage-tropic strain, and WSU5, a strain that is fibroblast tropic and can cause disease. Finally, upon necropsy of a WSU5-infected horse 4 years postinfection, EIAV-positive endothelial cells were detected in outgrowths of renal artery cultures. These findings identify a new cell type that is infectible with EIAV. The role of endothelial cell infection in the course of equine infectious anemia is currently unknown, but endothelial cell infection may be involved in the edema that can be associated with infection. Furthermore, the ability of EIAV to persistently infect endothelial cultures and the presence of virus in endothelial cells from a long-term carrier suggest that this cell type can serve as a reservoir for the virus during subclinical phases of infection.

Altered expression of extracellular matrix in human-cytomegalovirus-infected cells and a human artery organ culture model to study its biological relevance.

The influence of human cytomegalovirus (HCMV) on the transcription of 11 selected, representative extracellular matrix genes was investigated in cell culture. Northern blot hybridization indicated the downregulation of all mRNAs investigated. Based on our results and the known repression of other extracellular matrix transcripts and the beta-actin transcription during HCMV infection, we suggest that one molecular mechanism contributing to the cytopathic effect may be the transcriptional downregulation of genes encoding proteins involved in cell structure and intercellular connection. To further study the biological relevance of this and other pathogenetic mechanisms, we established a human renal artery organ culture system and characterized this new infection model for HCMV. Our model is a new suitable system for the investigation of molecular as well as functional consequences of HCMV infection in a more physiological microenvironment.