NADH oxidase is an important producer of ROS in vivo, and SIRT1 can inhibit production of an upstream signal, TNF-a, through deacetylation and inhibition of nuclear factor -kB in macrophages. By contrast, the pharmacologic inhibition of sirtuins has also been reported to inhibit the activation of the NFkB pathway and decrease the production of LPS-induced cytokines in J774 macrophages in vitro. Our TNF-a stimulation experiments in VSMCs yielded no hint of SIRT1 modulation, in contrast to findings by Zhang and coworkers. The discrepancy may be due to variability in cell culture conditions and/or TNF-a biostriking lead improvement lv ef long term activity compared with those of Zhang et al. It is however possible that other inflammatory stimuli combined with TNF-a induce upregulation of SIRT1, which may be part of a protective response of VSMCs to inflammatory events. In agreement with previous studies in human endothelial cells and STZ-diabetic mice, SIRT1 accumulation fell in diabetic compared to normoglycemic VSMCs, as confirmed using two different experimental procedures, and our in vitro high glucose studies are consistent with this pattern. To the best of our knowledge, this is the first demonstration of reduced SIRT1 levels in aortic VSMCs from STZ-diabetic rats. Because SIRT1 requires NAD for its enzymatic activity, a decline in NAD biosynthesis on high glucose or hyperglycemia may result in a significant reduction of SIRT1 levels and subsequent loss of the glucose- or insulinresponsive phenotypes. Accordingly, a significant reduction in NAD+ levels and SIRT1 activity in physiologically aged female Wistar rats has been recently reported. Similarly, although estrogen has been shown to inhibit VSMC proliferation under normal glucose concentrations, high glucose conditions abolish the antiproliferative effect of estrogen through as yet unknown mechanisms. In conclusion, the present study shows that previous diabetes induction in vivo negatively regulated SIRT1 amounts in rat VSMCs, consistent with the in vitro effects of high glucose concentrations, whereas we were unable to demonstrate SIRT1 modulation by TNF-a. In addition, 17b-estradiol decreased SIRT1 levels in VSMCs from normoglycemic but not diabetic rats most likely through ERa signaling, possibly as a downstream effect of AMPK activation. This negative regulation of SIRT1 could also be observed in human cell types, namely PBMCs freshly isolated from buffy coat, suggesting that findings in the rodent cell model can be translated to human settings. Whether the observed changes in SIRT1 protein levels in VSMCs are paralleled by changes in protein deacetylase activity remains to be determined. Future studies should also explore how the different levels of the SIRT1 regulatory network are connected in response to diverse cellular stressors, and what balances the net SIRT1 activity. As for the limited studies reporting the efficacy of SBRT for PVTT, the median OS was only 6�C8 months for fewer than 10 patients.