Background: Endothelial activation characterized by a pro-inflammatory phenotype following exposure to different stressors is commonly observed in cardiovascular disease. Forkhead (FOXO) transcription factors are important stress-activated regulators of cell cycle, cell differentiation and viability. Recently, it was reported that a gain of function (i.e., activating) single nucleotide polymorphism (SNP) in the FOXO3 gene exerts protective effects in chronic inflammatory disease. Moreover, cross talk with pro-inflammatory NFkB has been described. Acetylsalicylic acid (ASA) and Metformin, medications commonly used in clinical practice were shown to reduce inflammatory activation. Both reportedly activate the AMP-activated protein kinase (AMPK), a known upstream regulator of the transcription factor FOXO3. The aim of this study is to characterize intracellular signaling in endothelial cells following stress exposure and to determine possible anti-inflammatory effects of the ASA/Metformin-AMPK-FOXO3 signaling axis.

Methods: Studies were performed in Human Aortic Endothelial cells (HAECs) in vitro. Adenoviral vectors encoding the wild-type (WT-FOX3) and a constitutively active form of FOXO3 (TM-FOXO3) were used for gain and loss of function analysis, respectively. A pro-inflammatory phenotype was induced in HAECs by stimulation with LPS or H₂O₂. Activation of AMPK, FOXO3 and NFkB was determined by western blot. Target gene expression was quantified by qRT-PCR. Cellular redox stress levels were detected by commercially available DCFDHA and CellROX assays.

Results: Both Salicylate and Metformin, were able to activate FOXO3 downstream of AMPK by phosphorylation of AMPK-specific phosporylation sites. Immunostaining for FOXO3 showed no effect on cellular distribution of the transcription factor. In LPS-stimulated cells treated with 20mM Salicylate or Metformin induced a significant inhibition of pro-inflammatory markers such as ICAM-1 and VCAM-1. This effect was mediated by inhibition of IkB-α phosphorylation leading to attenuation of NFkB activity. However, activation or overexpression of FOXO3 did not alter NFkB dependent gene expression, indicating a lack of involvement of FOXO3 in the NFkB pro-inflammatory pathway. Moreover, Salicylate and Metformin significantly reduced ROS accumulation in H₂O₂ stimulated HAECs in a strictly concentration dependent manner.  This effect was AMPK dependent. Manipulation of AMPK activity by AICAR or compound C (activator and inhibitor, respectively) was accompanied by concurrent changes in FOXO3 activation. Moreover, FOXO3 overexpression significantly diminished cellularredox stress.

Conclusion: Salicylate and Metformin exert anti-inflammatory effects in endothelial cells by inhibition of NFkB signaling. Moreover, both drugs diminish cellular redox stress via AMPK-FOXO3 signaling. Further in vivo studies in models of cardiovascular injury are warranted to further elucidate the importance of these findings.