Because cytomegalovirus (CMV) may contribute to restenosis and atherosclerosis, and because smooth muscle cells (SMCs) are involved in these disease processes, we examined CMV/SMC interactions. Using confocal microscopy to identify a redox sensitive fluorescent marker, we found that CMV infection of SMCs generates intracellular reactive oxygen intermediates (ROIs). CMV also activated the cellular transcription factor NFkappaB, as demonstrated by increased NFkappaB binding to DNA (electrophoretic mobility shift assay). Antioxidants inhibited activation, suggesting a role of ROIs in CMV-induced NFkappaB activation. By using antioxidants to assess the role of ROIs in modulating virally-mediated effects, we also found CMV-induced ROIs: 1)are critical to the transactivation of the viral major immediate promoter (MIEP) by its immediate early protein IE72 (determined by cotransfection of an IE72 expression vector and a reporter gene downstream of tche MIEP); 2) are necessary for IE72 expression (determined by immunocytochemistry) and viral replication (determined by viral titer assay on indicator cells) following CMV infection of SMCs. We also demonstrated that aspirin (which has antioxidant and other activities) inhibits HCMV-induced ROS generation, HCMV-induced NFkappaB activation, IE72 expression, and HCMV replication. We further determined that CMV infection induces the prooxidant cyclooxygenase-2 (COX-2), by assaying for prostaglandin E2 [PGE2] release into the supernatant. We found expression of COX-2 protein (by Western blot) in infected cells but not in uninfected cells. Finally, we demonstrated that the immediate early viral proteins IE72 and IE84 transcriptionally activate the COX-2 promoter, by contransfecting smooth muscle cells with a COX-2 reporter gene and IE72 and IE84 expression vectors. Project finished. Manuscript published: Speir E, Yu Z.X, Ferrans V.F, Huang E.S., Epstein S.E. (1998)Aspirin attenuates cytomegalovirus infectivity and gene expression mediated by cyclo-oxygenase-2 in coronary artery smooth muscle cells. Circ Res, 83:210-216. - cytomegalovirus (CMV), antioxidants, reactive oxygen species (ROS)

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Intramural Research (Z01)
Project #
1Z01HL004974-05
Application #
6290449
Study Section
Cell Biology Integrated Review Group (CB)
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
1999
Total Cost
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
City
State
Country
United States
Zip Code