title: 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.
authors.affiliation: Department of Laboratory Diagnostics, Pomeranian Medical University, Szczecin, Poland.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Milkiewicz
authors.forename: Emilie
authors.initials: E
authors.lastname: Roudier
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Doyle
authors.forename: Anastassia
authors.initials: A
authors.lastname: Trifonova
authors.forename: Olivier
authors.initials: O
authors.lastname: Birot
authors.forename: Tara L
authors.initials: TL
authors.lastname: Haas
mesh_terms: D020533:Angiogenesis Inhibitors / Q000378:metabolism*; D000818:Animals; D002196:Capillaries / Q000378:metabolism / Q000473:pathology; D002453:Cell Cycle; D015687:Cell Hypoxia; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D051858:Forkhead Transcription Factors / Q000378:metabolism*; D006614:Hindlimb / Q000098:blood supply / Q000473:pathology; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D007511:Ischemia / Q000378:metabolism* / Q000473:pathology; D008297:Male; D009132:Muscles / Q000098:blood supply* / Q000378:metabolism / Q000473:pathology*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D018384:Oxidative Stress; D010766:Phosphorylation; D011485:Protein Binding; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D013314:Stress, Mechanical; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D000071161:Forkhead Box Protein O1
chemical_list: D020533:Angiogenesis Inhibitors; D048049:Extracellular Signal-Regulated MAP Kinases; D051858:Forkhead Transcription Factors; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009419:Nerve Tissue Proteins; D051057:Proto-Oncogene Proteins c-akt; D051736:Proto-Oncogene Proteins c-mdm2; D042461:Vascular Endothelial Growth Factor A; C074639:Foxo1 protein, rat; D000071161:Forkhead Box Protein O1; C497208:Mdm2 protein, rat