Abstract 5646: Endothelial Intrinsic NF-κB Signaling Regulates iNOS And eNOS Expression in vivo in Endotoxemic Mice
We have previously demonstrated that endothelial NF-κB blockade mitigates septic shock and septic vascular dysfunction (Circulation 118: S570, 2008). Since endothelial and inducible nitric oxide synthase (iNOS and eNOS) are known to be involved in vascular dysfunction, this study examined the contribution of endothelial intrinsic NF-κB signaling in LPS induced alterations in iNOS and eNOS expression. Wild type (WT) and transgenic mice (TG) that conditionally overexpress a mutant I-κBα selectively on endothelium were injected with saline (1 ml/kg, i.p.) or E Coli LPS (10 mg/kg, i.p.). Aortic levels of iNOS and eNOS proteins were determined by Western blot. The iNOS and eNOS bands were quantified using densitometry and expressed as arbitrary optical density units. Plasma and aortic levels of nitrite/nitrate, and aortic level of TNF-α were measured using commercial kits. Aortic level of iNOS protein (O.D units) for WT-Con, TG-Con, WT-LPS and TG-LPS groups were 0±0, 0±0, 935±107 and 257±58 (p < 0.01, WT-LPS vs other 3 groups). Plasma and aortic levels of nitrite/nitrate, for WT-Con, TG-Con, WT-LPS and TG-LPS groups, were 4.4±0.7, 3.9±0.4, 19.3±3.4 and 6.9±1.3 μM in plasma, and 5.8±1.9, 5.8±0.9, 40.2±3.9 and 19.9±2.0 nmole/mg protein in aorta (p < 0.01, WT-LPS vs other 3 groups). Aortic level of eNOS protein (O.D units) for WT-Con, TG-Con, WT-LPS and TG-LPS groups were 896±54, 799±61, 586±46 and 838±49 (p < 0.05, WT-LPS vs other 3 groups). Down-regulation of eNOS expression was associated with increased aortic TNF-α expression. Aortic levels of TNF-α (pg/mg protein) for WT-Con, TG-Con, WT-LPS and TG-LPS groups were 68±26, 23±12, 753±132 and 369±54 (p < 0.01, WT-LPS vs other 3 groups). IHC staining of aortic sections showed that TG-LPS mice had a significantly reduction in LPS-induced iNOS or TNF-α expression in both endothelial and smooth muscle cells, indicating a paracrine interaction between the two cells. These results suggest blockade of endothelial NF-κB pathway mitigates septic vascular dysfunction by inhibiting vascular iNOS expression and by preventing eNOS down-regulation.
(Supported by NIH GM063907)