Gene expression profiling of endothelial cells derived from Foxo1(+/+) and Foxo1(-/-) ES cells in response to VEGF
ABSTRACT: VEGF induces elongation of endothelial cells (ECs) that are derived from wild-type (Foxo1(+/+)) ES cells. VEGF-induced EC elongation is an important process of angiogenesis. ECs derived from Foxo1(-/-) ES cells fail to elongate in response to VEGF. Gene expression profiling of wild-type and Foxo1(-/-) ECs in the presence or absence of VEGF stimulation identifies responsible genes that regulate EC elongation. One wild-type (Foxo1(+/+)) ES cell clone and one Foxo1(-/-) ES cell clone were used. ES cells were cultured on OP9 stromal cell layer in the presence or absence of VEGF (10 ng/ml). After 6.5 days, VE-cadherin+ CD31+ ECs were isolated by FACS and used for total RNA extraction. Three independent experiments were performed for each condition.
Project description:Endothelial cells (ECs) are plastic cells that can switch between growth states with different bioenergetic and biosynthetic requirements. Although quiescent in most healthy tissues, ECs divide and migrate rapidly upon proangiogenic stimulation. Adjusting endothelial metabolism to the growth state is central to normal vessel growth and function, yet it is poorly understood at the molecular level. Here we report that the forkhead box O (FOXO) transcription factor FOXO1 is an essential regulator of vascular growth that couples metabolic and proliferative activities in ECs. Endothelial-restricted deletion of FOXO1 in mice induces a profound increase in EC proliferation that interferes with coordinated sprouting, thereby causing hyperplasia and vessel enlargement. Conversely, forced expression of FOXO1 restricts vascular expansion and leads to vessel thinning and hypobranching. We find that FOXO1 acts as a gatekeeper of endothelial quiescence, which decelerates metabolic activity by reducing glycolysis and mitochondrial respiration. Mechanistically, FOXO1 suppresses signalling by MYC (also known as c-MYC), a powerful driver of anabolic metabolism and growth. MYC ablation impairs glycolysis, mitochondrial function and proliferation of ECs while its EC-specific overexpression fuels these processes. Moreover, restoration of MYC signalling in FOXO1-overexpressing endothelium normalizes metabolic activity and branching behaviour. Our findings identify FOXO1 as a critical rheostat of vascular expansion and define the FOXO1-MYC transcriptional network as a novel metabolic checkpoint during endothelial growth and proliferation.
Project description:Forkhead box-O transcription factors (FoxOs) transduce a wide range of extracellular signals, resulting in changes in cell survival, cell cycle progression, and several cell type-specific responses. FoxO1 is expressed in many cell types, including endothelial cells (ECs). Previous studies have shown that Foxo1 knockout in mice results in embryonic lethality at E11 because of impaired vascular development. In contrast, somatic deletion of Foxo1 is associated with hyperproliferation of ECs. Thus, the precise role of FoxO1 in the endothelium remains enigmatic.To determine the effect of endothelial-specific knockout and overexpression of FoxO1 on vascular homeostasis.We show that EC-specific disruption of Foxo1 in mice phenocopies the full knockout. Although endothelial expression of FoxO1 rescued otherwise Foxo1-null animals, overexpression of constitutively active FoxO1 resulted in increased EC size, occlusion of capillaries, elevated peripheral resistance, heart failure, and death. Knockdown of FoxO1 in ECs resulted in marked inhibition of basal and vascular endothelial growth factor-induced Akt-mammalian target of rapamycin complex 1 (mTORC1) signaling.Our findings suggest that in mice, endothelial expression of FoxO1 is both necessary and sufficient for embryonic development. Moreover, FoxO1-mediated feedback activation of Akt maintains growth factor responsive Akt/mTORC1 activity within a homeostatic range.
Project description:The REG?-proteasome serves as a short-cut for the destruction of certain intact mammalian proteins in the absence of ubiquitin- and ATP. The biological roles of the proteasome activator REG? are not completely understood. Here we demonstrate that REG? controls degradation of protein kinase A catalytic subunit-? (PKAca) both in primary human umbilical vein endothelial cells (HUVECs) and mouse embryonic fibroblast cells (MEFs). Accumulation of PKAca in REG?-deficient HUVECs or MEFs results in phosphorylation and nuclear exclusion of the transcription factor FoxO1, indicating that REG? is involved in preserving FoxO1 transcriptional activity. Consequently, VEGF-induced expression of the FoxO1 responsive genes, VCAM-1 and E-Selectin, was tightly controlled by REG? in a PKA dependent manner. Functionally, REG? is crucial for the migration of HUVECs. REG?(-/-) mice display compromised VEGF-instigated neovascularization in cornea and aortic ring models. Implanted matrigel plugs containing VEGF in REG?(-/-) mice induced fewer capillaries than in REG?(+/+) littermates. Taken together, our study identifies REG? as a novel angiogenic factor that plays an important role in VEGF-induced expression of VCAM-1 and E-Selectin by antagonizing PKA signaling. Identification of the REG?-PKA-FoxO1 pathway in endothelial cells (ECs) provides another potential target for therapeutic intervention in vascular diseases.
Project description:Loss of the microvascular (MV) network results in tissue ischemia, loss of tissue function, and is a hallmark of chronic diseases. The incorporation of a functional vascular network with that of the host remains a challenge to utilizing engineered tissues in clinically relevant therapies. We showed that vascular-bed-specific endothelial cells (ECs) exhibit differing angiogenic capacities, with kidney microvascular endothelial cells (MVECs) being the most deficient, and sought to explore the underlying mechanism. Constitutive activation of the phosphatase PTEN in kidney MVECs resulted in impaired PI3K/AKT activity in response to vascular endothelial growth factor (VEGF). Suppression of PTEN in vivo resulted in microvascular regeneration, but was insufficient to improve tissue function. Promoter analysis of the differentially regulated genes in KMVECs suggests that the transcription factor FOXO1 is highly active and RNAseq analysis revealed that hyperactive FOXO1 inhibits VEGF-Notch-dependent tip-cell formation by direct and indirect inhibition of DLL4 expression in response to VEGF. Inhibition of FOXO1 enhanced angiogenesis in human bio-engineered capillaries, and resulted in microvascular regeneration and improved function in mouse models of injury-repair.
Project description:Analysis of transcriptional changes upon silencing of endothelial VEGF. Because transcription factor Foxo1 levels increase under VEGF silencing, we hypothesized that Foxo1 transcriptional activity would be apparent in a whole-genome microarray. Double-silencing of VEGF+Foxo1 was also performed to determine which transcriptional changes are Foxo1-dependent in a VEGF-deficient background.
Project description:The Ewing sarcoma (ES) EWS-FLI1 chimeric oncoprotein is a prototypic aberrant ETS transcription factor with activating and repressive regulatory functions. We report that EWS-FLI1-repressed promoters are enriched in forkhead box (FOX) recognition motifs, and identify FOXO1 as a EWS-FLI1-suppressed regulator orchestrating a major subset of EWS-FLI1-repressed genes. In addition to FOXO1 regulation by direct promoter binding of EWS-FLI1, its subcellular localization and activity is regulated by cyclin-dependent kinase 2- and AKT-mediated phosphorylation downstream of EWS-FLI1. Restoration of nuclear FOXO1 expression in ES cells impaired proliferation and significantly reduced clonogenicity. Gene-expression profiling revealed a significant overlap between EWS-FLI1-repressed and FOXO1-activated genes. As a proof of principle for a potential therapeutic application of our findings, the treatment of ES cell lines with methylseleninic acid (MSA) reactivated endogenous FOXO1 in the presence of EWS-FLI1 in a dose- and time-dependent manner and induced massive cell death dependent on FOXO1. In an orthotopic xenograft mouse model, MSA increased FOXO1 expression in the tumor paralleled by a significant decrease in ES tumor growth. FOXO1 reactivation by small molecules may therefore serve as a promising strategy for a future ES-specific therapy.
Project description:BACKGROUND:Venous malformations (VMs), most of which associated with activating mutations in the endothelial cells (ECs) tyrosine kinase receptor TIE2, are characterized by dilated and immature veins with scarce smooth muscle cells (SMCs) coverage. However, the underlying mechanism of interaction between ECs and SMCs responsible for VMs has not been fully understood. METHODS:Here, we screened 5 patients with TIE2-L914F mutation who were diagnosed with VMs by SNP sequencing, and we compared the expression of platelet-derived growth factor beta (PDGFB) and ?-SMA in TIE2 mutant veins and normal veins by immunohistochemistry. In vitro, we generated TIE2-L914F-expressing human umbilical vein endothelial cells (HUVECs) and performed BrdU, CCK-8, transwell and tube formation experiments on none-transfected and transfected ECs. Then we investigated the effects of rapamycin (RAPA) on cellular characteristics. Next we established a co-culture system and investigated the role of AKT/FOXO1/PDGFB in regulating cross-talking of mutant ECs and SMCs. RESULTS:VMs with TIE2-L914F mutation showed lower expression of PDGFB and ?-SMA than normal veins. TIE2 mutant ECs revealed enhanced cell viability and motility, and decreased tube formation, whereas these phenotypes could be reversed by rapamycin. Mechanically, RAPA ameliorated the physiological function of mutant ECs by inhibiting AKT-mTOR pathway, but also facilitated the nuclear location of FOXO1 and the expression of PDGFB in mutant ECs, and then improved paracrine interactions between ECs and SMCs. Moreover, TIE2 mutant ECs strongly accelerated the transition of SMCs from contractile phenotype to synthetic phenotype, whereas RAPA could prevent the phenotype transition of SMCs. CONCLUSIONS:Our data demonstrate a previously unknown mechanistic linkage of AKT-mTOR/FOXO1 pathway between mutant ECs and SMCs in modulating venous dysmorphogenesis, and AKT/FOXO1 axis might be a potential therapeutic target for the recovery of TIE2-mutation causing VMs. Video Abstract.
Project description:Nucleoside diphosphate kinase B (NDPK-B) acts as a protective factor in the retinal vasculature. NDPK-B deficiency leads to retinal vasoregression mimicking diabetic retinopathy (DR). Angiopoetin 2 (Ang-2), an initiator of retinal vasoregression in DR, is upregulated in NDPK-B deficient retinas and in NDPK-B depleted endothelial cells (ECs) in vitro. We therefore investigated the importance of Ang-2 in NDPK-B deficient retinas and characterized the mechanisms of Ang-2 upregulation upon NDPK-B depletion in cultured ECs. The crucial role of retinal Ang-2 in the initiation of vasoregression was verified by crossing NDPK-B deficient with Ang-2 haplodeficient mice. On the molecular level, FoxO1, a transcription factor regulating Ang-2, was upregulated in NDPK-B depleted ECs. Knockdown of FoxO1 abolished the elevation of Ang-2 induced by NDPK-B depletion. Furthermore O-GlcNAcylated FoxO1 was found preferentially in the nucleus. An increased O-GlcNAcylation of FoxO1 was revealed upon NDPK-B depletion. In accordance, the inhibition of protein O-GlcNAcylation normalized NDPK-B depletion induced Ang-2 upregulation. In summary, we demonstrated that the upregulation of Ang-2 upon NDPK-B deficiency is driven by O-GlcNAcylation of FoxO1. Our data provide evidence for a central role of protein O-GlcNAcylation in NDPK-B associated vascular damage and point to the hexosamine pathway as an important target in retinal vasoregression.
Project description:Poly(ADP-ribose) polymerase 1 (PARP1) regulates gene transcription in addition to functioning as a DNA repair factor. Forkhead box O1 (FoxO1) is a transcription factor involved in extensive biological processes. Here, we report that PARP1 binds to two separate motifs on the FoxO1 promoter and represses its transcription in a polymerase-independent manner. Using PARP1-knock out (KO) cells, wild-type-PARP1-complemented cells and catalytic mutant PARP1E988K-reconstituted cells, we investigated transcriptional regulation by PARP1. PARP1 loss led to reduced DNA damage response and ~362-fold resistance to five PARP inhibitors (PARPis) in Ewing sarcoma cells. RNA sequencing showed 492 differentially expressed genes in a PARP1-KO subline, in which the FoxO1 mRNA levels increased up to more than five times. The change in the FoxO1 expression was confirmed at both mRNA and protein levels in different PARP1-KO and complemented cells. Moreover, exogenous PARP1 overexpression reduced the endogenous FoxO1 protein in RD-ES cells. Competitive EMSA and ChIP assays revealed that PARP1 specifically bound to the FoxO1 promoter. DNase I footprinting, mutation analyses, and DNA pulldown FREP assays showed that PARP1 bound to two particular nucleotide sequences separately located at -813 to -826?bp and -1805 to -1828?bp regions on the FoxO1 promoter. Either the PARPi olaparib or the PARP1 catalytic mutation (E988K) did not impair the repression of PARP1 on the FoxO1 expression. Exogenous FoxO1 overexpression did not impair cellular PARPi sensitivity. These findings demonstrate a new PARP1-gene promoter binding mode and a new transcriptional FoxO1 gene repressor.
Project description:Forkhead box O 1 (Foxo1) controls the expression of proteins that carry out processes leading to skeletal muscle atrophy, making Foxo1 of therapeutic interest in conditions of muscle wasting. The transcription of Foxo1-regulated proteins is dependent on the translocation of Foxo1 to the nucleus, which can be repressed by insulin-like growth factor-1 (IGF-1) treatment. The role of Foxo1 in muscle atrophy has been explored at length, but whether Foxo1 nuclear activity affects skeletal muscle excitation-contraction (EC) coupling has not yet been examined. Here, we use cultured adult mouse skeletal muscle fibers to investigate the effects of Foxo1 overexpression on EC coupling. Fibers expressing Foxo1-green fluorescent protein (GFP) exhibit an inability to contract, impaired propagation of action potentials, and ablation of calcium transients in response to electrical stimulation compared with fibers expressing GFP alone. Evaluation of the transverse (T)-tubule system morphology, the membranous system involved in the radial propagation of the action potential, revealed an intact T-tubule network in fibers overexpressing Foxo1-GFP. Interestingly, long-term IGF-1 treatment of Foxo1-GFP fibers, which maintains Foxo1-GFP outside the nucleus, prevented the loss of normal calcium transients, indicating that Foxo1 translocation and the atrogenes it regulates affect the expression of proteins involved in the generation and/or propagation of action potentials. A reduction in the sodium channel Nav1.4 expression in fibers overexpressing Foxo1-GFP was also observed in the absence of IGF-1. We conclude that increased nuclear activity of Foxo1 prevents the normal muscle responses to electrical stimulation and that this indicates a novel capability of Foxo1 to disable the functional activity of skeletal muscle.