Cytomegalovirus beta2.7 RNA transcript protects endothelial cells against apoptosis during ischemia/reperfusion injury.

Background: Ischemia/reperfusion (I/R) injury causes endothelial cell (EC) dysfunction and can precipitate apoptosis. Complex I of the mitochondrial respiratory chain is a target for I/R injury. The beta2.7 RNA transcript encoded by human cytomegalovirus (CMV) has been shown to stabilize Complex I by direct physical interaction. In this study, we investigated whether stabilizing Complex I in EC in an in vitro model of ischemia could prevent apoptosis.

Methods: Lentiviral vectors expressing a full-length beta2.7 RNA were generated from a human immunodeficiency virus-1 (HIV-1) construct, in which the viral promoter had been inactivated and virtually all the viral accessory proteins deleted in order to maximize safety. beta2.7 gene expression in transduced endothelial cells was examined by reverse transcript-polymerase chain reaction (RT-PCR). EC were prepared from rat aorta. An in vitro hypoxia/reperfusion (H/R) and I/R injury models were set up and apoptosis was assessed using caspase-3 activity. Reactive oxygen species (ROS) production in the endothelial cells was assessed by the capacity to oxidize non-fluorescent dihydrorhodamine-123 (DHR-123) to fluorescent rhodamine-123 and measured by flow cytometry.

Results: H/R and I/R injury induced formation of ROS and EC apoptosis. Overexpression of the viral beta2.7 RNA, which stabilizes Complex I, reduced ROS production and inhibited EC apoptosis.

Conclusions: beta2.7 RNA is a novel effector molecule that can protect rat aortic endothelial cells from I/R injury causing apoptosis. As a non-coding RNA, beta2.7 RNA will not induce an immune response in the recipient. We have shown that overexpression of beta2.7 RNA can protect RAEC from H/R- or I/R-mediated apoptosis by reduction of ROS formation.