Lentiviral-mediated overexpression of Bcl-xL protects primary endothelial cells from ischemia/reperfusion injury-induced apoptosis.

Background: Endothelial cells (EC) respond to mild injurious stimuli by upregulating anti-apoptotic gene expression to maintain endothelial integrity. EC dysfunction and apoptosis resulting from ischemia/reperfusion injury may contribute to chronic allograft rejection. We optimized conditions for lentiviral vector (LVV) transduction of rat aortic endothelial cells (RAEC) and investigated whether LVV delivery of the anti-apoptotic gene, Bcl-xL, protects RAEC from apoptotic death using in vitro models of hypoxia and ischemia/reperfusion injury.

Methods: LVV containing Bcl-xL were generated from a human immunodeficiency virus (HIV)-1 construct. EC were prepared from rat aorta. Hypoxia/reperfusion (H/R) or ischemia/reperfusion (I/R) injury was induced in vitro and apoptosis was assessed using caspase-3 activity, Annexin V/PI and TUNEL staining.

Results: After in vitro induction of H/R or I/R injury, RAEC showed duration-dependent apoptosis. We confirmed the damaging effect of the reperfusion phase. Endogenous Bax expression increased with I/R injury, whereas endogenous Bcl-xL remained constant. RAEC transduced with LVV expressing Bcl-xL were protected from early apoptosis caused by I/R injury, correlating with reduced cytochrome c release into the cytosol.

Conclusions: Overexpressing Bcl-xL protects RAEC from I/R injury. This protective effect may be attributed to altering the balance of pro- and anti-apoptotic proteins, resulting in sequestration of the harmful Bax protein, and may open up new strategies for controlling chronic allograft rejection.