Attenuated acute cardiac rejection in NOS2 -/- recipients correlates with reduced apoptosis

Abstract

Background - The mechanisms through which NOS2-mediated pathways regulate graft failure in acute cardiac rejection are ill defined. To determine whether apoptosis promoted by NOS2 may contribute, we used a heterotopic transplant model to study mouse cardiac allografts placed in recipients with targeted gene deletion of NOS2. Methods and Results - Using 5 different indexes of apoptosis, we showed that mouse cardiac allografts placed in NOS2 -/- recipients (n = 7) had reduced apoptotic activity compared with those in NOS2 +/+ controls (n = 8). There were significantly fewer TUNEL-positive nuclei per high-powered field (P < 0.01), less DNA fragmentation (antinucleosome ELISA; P < 0.05), lower corrected transcript levels for caspase-1 and -3 (32P reverse transcriptase-polymerase chain reaction; P < 0.01), and reduced caspase-3 activity (cleavage of DEVD-pNA [P < 0.001] and poly [ADP-ribose] polymerase) in grafts from NOS2 -/- recipients. This concordant reduction in apoptotic indexes paralleled the improved histological outcome of grafts transplanted into NOS2 -/- recipients (assessed as rejection scores; P = 0.012). To identify pathways controlled by NOS2, we compared intragraft transcript levels of potential triggers and regulators. Whereas Fas ligand/Fas and tumor necrosis factor (TNF)-α/TNF receptor-1 levels were not altered by NOS2 deficiency, transcript levels for p53 were significantly lower in grafts from NOS2 -/- recipients, coinciding with a significant increase in the antiapoptotic Bcl-2/Bax balance and decrease in Bcl-X1 levels. Conclusions - Using NOS2 knockout mice, we demonstrated that NOS2-mediated pathways can promote acute rejection, at least in part, by inducing apoptotic cell death. When NOS2 is present, p53 might control NOS2-mediated apoptosis by stimulating Bax and repressing Bcl- 2 and Bcl-X1 expression, which may activate the cell death program n the rejecting heart.