The M33 gene of murine cytomegalovirus (MCMV) is involved in the stimulation of VEGF-A production by mouse macrophages

Abstract

Purpose: Our laboratory recently demonstrated that chronic MCMV infection of healthy C57BL/6 mice results in a more severe experimental choroidal neovascularization (CNV) (PLoS Pathogens 8:e1002671, 2012). Because the human cytomegalovirus (HCMV)-encoded chemokine receptor US28 has been shown to play a multifunctional role in angiogenesis, we hypothesized that the M33 gene of MCMV (the MCMV homologue to HCMV US28) plays a similar role in experimental CNV. The purpose of this study is to test the hypothesis that the M33 gene of MCMV is involved in the stimulation of VEGF production by mouse macrophages.

Methods: Monolayers of IC-21 mouse macrophages were treated for 24 hours with either M33 siRNAs (single or combined M33-1 siRNA and M33-2 siRNA) or scrambled siRNAs (control) using either HiPerFect transfection reagent (Qiagen) or Lipofectamine RNAiMAX transfection reagent (Invitrogen) and inoculated with MCMV (moi=3 pfu/cell). All siRNA-treated and control MCMV-infected monolayers were harvested at 24 and 48 hours post-infection and subjected to western blot analysis for the comparison of VEGF-A protein production.

Results: Although no significant differences were observed when comparing relative band densities of VEGF-A production by siRNA-treated MCMV-infected macrophages at 24-hour post-infection, treatment using a combination of M33-1/M33-2 siRNAs resulted in a decrease in VEGF-A protein production by ~51%. Use of Lipofectamine RNAiMAX transfection reagent was superior to HiPerFect transfection reagent in reducing VEGF-A protein production by the M33 siRNAs.

Conclusions: The M33 gene of MCMV is involved in the stimulation of VEGF-A production by IC-21 mouse macrophages, but the downregulation of M33 gene by siRNAs did not result in a greater reduction in VEGF-A protein production as expected. We therefore postulate that one or more genes in addition to the M33 gene of MCMV contribute to the stimulation of VEGF-A production by mouse macrophages.

Keywords: 492 cytomegalovirus • 748 vascular endothelial growth factor • 412 age-related macular degeneration