Identification of a Mechanism Underlying Regulation of the Anti-Angiogenic Forkhead Transcription Factor FoxO1 in Cultured Endothelial Cells and Ischemic Muscle

Abstract

Chronic limb ischemia, a complication commonly observed in conjunction with cardiovascular disease, is characterized by insufficient neovascularization despite the up-regulation of pro-angiogenic mediators. One hypothesis is that ischemia induces inhibitory signals that circumvent the normal capillary growth response. FoxO transcription factors exert anti-proliferative and pro-apoptotic effects on many cell types. We studied the regulation of FoxO1 protein in ischemic rat skeletal muscle following iliac artery ligation and in cultured endothelial cells. We found that FoxO1 expression was increased in capillaries within ischemic muscles compared with those from rats that underwent a sham operation. This finding correlated with increased expression of p27(Kip1) and reduced expression of Cyclin D1. Phosphorylated Akt was reduced concurrently with the increase in FoxO1 protein. In skeletal muscle endothelial cells, nutrient stress as well as lack of shear stress stabilized FoxO1 protein, whereas shear stress induced FoxO1 degradation. Endogenous FoxO1 co-precipitated with the E3 ubiquitin ligase murine double minute-2 (Mdm2) in endothelial cells, and this interaction varied in direct relation to the extent of Akt and Mdm2 phosphorylation. Moreover, ischemic muscles had a decreased level of Mdm2 phosphorylation and a reduced interaction between Mdm2 and FoxO1. Our results provide novel evidence that the Akt-Mdm2 pathway acts to regulate endothelial cell FoxO1 expression and illustrate a potential mechanism underlying the pathophysiological up-regulation of FoxO1 under ischemic conditions.

FoxO3a and disease progression

“…Intriguingly, knockout or knockdown of MDM2 alone increases FoxO3a protein levels. This effect was shown to be mediated by MDM2-induced polyubiquitination of FoxO proteins [27,28] , whereas another study showed that MDM2 catalyzes multiple monoubiquitination of FoxO4 rather than polyubiquitination [28] . When FoxO3a is located to the cytoplasm by Akt, FoxO3a becomes ubiquitinated and this event triggers a proteasomedependent degradation process.…”

“…The single molecule RING-finger E3 ligase murine double minute 2 (MDM2) promotes ubiquitination of FoxO3a as well as FoxO1 and FoxO4, facilitating their degradation [27] . Intriguingly, knockout or knockdown of MDM2 alone increases FoxO3a protein levels.…”

FOXOs and Sirtuins in Vascular Growth, Maintenance, and Aging

“…48 Further support for a critical role of FOXOs in vascular growth stems from the observation that FOXOs are upregulated in ECs of ischemic muscles and that FOXO3 deficiency enhances vessel density after ischemic stress in mice. 44,49 The cell responses and target genes controlled by different FOXOs in ECs are overlapping but not entirely redundant. 44,48,50 -52 For example, overexpression of a constitutively active FOXO1 or FOXO3 mutant inhibits endothelial sprouting and migration, whereas forced expression of FOXO4 has no discernible effect.…”

Resveratrol modulates the angiogenic response to exercise training in skeletal muscles of aged men

“…FoxO1 limits angiogenesis (38) in part by increasing angiostatic TSP-1 (56). FoxO1 protein levels were measured in the present muscle samples, but no effect of resveratrol supplementation on FoxO1 protein expression was observed.…”

“…In addition, SIRT-1 may promote angiogenesis by deacetylating and inhibiting the angiostatic transcription factor forkhead box protein O1 (FoxO1) (47). FoxO1 has been shown to limit angiogenesis in part via upregulation of TSP-1 (38,56). Resveratrol supplementation would, therefore, through these separate mechanisms, be expected to increase VEGF levels in skeletal muscle and promote angiogenesis.…”