Figure 7
From: eNAMPT neutralization reduces preclinical ARDS severity via rectified NFkB and Akt/mTORC2 signaling
eNAMPT-neutralizing strategies preserves mTORC2 and UCHL1 protein expression in rat/porcine LPS/VILI models. (A, B) Evaluation of levels of total mTOR and the mTORC2 protein, Rictor, in LPS/VILI-exposed rat (n = 3) and porcine lung homogenates (n = 3) demonstrated marked reductions in total mTOR and Rictor protein expression which are significantly rectified by the eNAMPT-neutralizing mAb quantified by densitometric analysis (*p < 0.05 control vs. untreated LPS/VILI; **p < 0.05 mAb LPS/VILI vs. untreated LPS/VILI). (C) Human pulmonary artery endothelial cells (EC) were transfected with UCHL1 siRNA (100 nM) GE Dharmacon, Lafayette, CO) or non-specific scrambled sequence using transfection reagent siPORT (Ambion, Austin, TX) as previously described9. Cell lysates prepared 72 h post transfection for western blotting studies revealed reduced levels of UCHL1 and AktS473 phosphorylation, quantified by densitometric analysis (*p < 0.05 control vs. UCHL1 siRNA). (D) UCHL1 protein expression was assessed in lung homogenates obtained from LPS/VILI-exposed Yucatan minipigs (n = 3) with and without treatment with the eNAMPT-neutralizing mAb. lung homogenates (n = 3) compared to control pig tissues. The LPS/VILI-mediated reduction in UCHL1 expression in lung tissues was restored to baseline levels by the eNAMPT mAb (*p < 0.05 control vs. untreated LPS/VILI; **p < 0.05 mAb LPS/VILI vs. untreated LPS/VILI).
