Signaling via PI3K/FOXO1A pathway modulates formation and survival of human embryonic stem cell-derived endothelial cells

Abstract

Vascular derivatives of human embryonic stem cells (hESC) are being developed as sources of tissue-specific cells for organ regeneration. However, identity of developmental pathways that modulate the specification of endothelial cells is not known yet. We studied phosphatidylinositol 3-kinase (PI3K)-Forkhead box O transcription factor 1A (FOXO1A) pathways during differentiation of hESC toward endothelial lineage and on proliferation, maturation, and cell death of hESC-derived endothelial cells (hESC-EC). During differentiation of hESC, expression of FOXO1A transcription factor was linked to the expression of a cluster of angiogenesis- and vascular remodeling-related genes. PI3K inhibitor LY294002 activated FOXO1A and induced formation of CD31(+) hESC-EC. In contrast, differentiating hESC with silenced FOXO1A by small interfering RNA (siRNA) showed lower mRNA levels of CD31 and angiopoietin2. LY294002 decreased proliferative activity of purified hESC-EC, while FOXO1A siRNA increased their proliferation. LY294002 inhibits migration and tube formation of hESC-EC; in contrast, FOXO1A siRNA increased in vitro tube formation activity of hESC-EC. After in vivo conditioning of cells in athymic nude rats, cells retain their low FOXO1A expression levels. PI3K/FOXO1A pathway is important for function and survival of hESC-EC and in the regulation of endothelial cell fate. Understanding these properties of hESC-EC may help in future applications for treatment of injured organs.

FIG. 1.

PI3K/FOXO1A signaling-related gene expression during human embryonic stem cells (hESC) differentiation. hESC were differentiated via embryoid body (EB) formation toward hESC-derived endothelial cells (hESC-EC). HUVEC were used as control endothelial cells. (A) Changes in mRNA levels of embryonic stem cell marker OCT4, CD31, angiogenesis markers angiopoietin2, and Tie2 at different steps of endothelial differentiation. Heat map with agglomerative clustering (B) and bar graphs (C) showing changes in mRNA levels of PI3K/FOXO elements during different steps of endothelial differentiation. Bar graphs in (B) showing differences in gene expression in HUVEC versus those in hESC-EC as a baseline. Data are expressed as fold changes versus mRNA levels in undifferentiated hESC; mean±SEM. Statistical significance was determined by one-way ANOVA. *P<0.05, **P<0.01, ***P<0.01, from three biological replicates, one-way ANOVA with Tukey post hoc test. (D) Schematic diagram of Ingenuity Pathway Analysis-mapped mechanistic interactions of VEGF, PTEN, FOXO1A, and various angiogenic modulator elements. Arrows between nodes represent direct (solid lines) and indirect (dashed lines) interactions between molecules as supported by information in the Ingenuity pathway knowledge base. ANOVA, analysis of variance; FOXO1A, Forkhead box O transcription factor 1A; HUVEC, human umbilical cord vein cells; ITGB1, integrin beta-1; PDGFRA, platelet-derived growth factor receptor alpha; PI3K, phosphatidylinositol 3-kinase; PIK3R2, phosphoinositide-3-kinase, regulatory subunit 2 beta; PRKCA, protein kinase C alpha; PRKCB, protein kinase C beta; PRKCZ, protein kinase C zeta; PTEN, phosphatase and tensin homolog; VEGF, vascular endothelial growth factor. Color images available online at www.liebertpub.com/scd

FIG. 2.

PI3K/FOXO1A signaling modulates endothelial differentiation of hESC. Bar graphs showing mRNA levels of CD31 (A), angiopoietin2 (B), and Tie2 (C) in differentiating hESC (EB) with LY294002 and FOXO1A siRNA. (D) Bar graph showing CD31⁺ hESC-EC population in the presence of FOXO1A siRNA or LY294002 (10 μM). mean±SEM, *P<0.05, **P<0.01, ***P<0.001 versus control group, three biological replicates, Student's t-test. siRNA, small interfering RNA.

FIG. 3.

PI3K/FOXO1A signaling modulates proliferative activity of hESC-EC. Bar graphs showing changes in mRNA levels of CD31, angiopoietin2, and Tie2 in differentiated hESC-EC in response to 24 h treatment with LY294002 (A–C) or FOXO1A siRNA (D–F). (G) Representative picture of Ki67⁺ cells in hESC-EC population (magnification: 20×). Changes in proliferation rate of the hESC-EC population (Ki67⁺ cells) after 24 h (H) and in colony formation activity (I, J) in response to LY294002 or FOXO1A siRNA treatments for 3 days. Data are expressed as fold changes versus percentage of Ki67⁺ cells in control hESC-EC group, mean±SEM. Fold changes in mRNA levels are shown versus hESC. *P<0.05, **P<0.01, ***P<0.001 versus control, n=3 biological replicates. Color images available online at www.liebertpub.com/scd

FIG. 4.

In vitro angiogenesis and in vivo conditioning of hESC-EC. (A) Representative Matrigel tube formation images of hESC-EC pretreated with LY294002 or FOXO1A siRNA. Cells were stained with vital dye TMRM (red). Bar graph showing average and total tube lengths and number of formed nodes after LY294002 (B–D) 24 h FOXO1A or nontargeting scrambled (NT) siRNA (E–G). *P<0.05, **P<0.01, ***P<0.001 versus control, n=3 biological replicates. (H) Representative image of histological section stained with hematoxylin-eosin showing subcutaneously engrafted hESC-EC. (I–K) Bar graphs showing FOXO1A, angiopoietin2 and Tie2 mRNA levels before and after in vivo conditioning of hESC-EC in Matrigel plugs transplanted into athymic nude rats. Data are mean±SEM [n=3–10 per group from two independent experiments (12 rats per group)]. Color images available online at www.liebertpub.com/scd

FIG. 5.

Mechanism of action and effects of modulation of PI3K-FOXO1A signaling pathway. Schematic summary shows that activation of FOXO1A with PI3K inhibitor LY294002 resulted in increased yield of endothelial differentiation (labeled as red panels). Proliferative and tube formation capacity decreased (labeled as blue panels) in the differentiated endothelial cells. Modulation of PI3K-FOXO1A signaling also altered angiopoietin2-Tie2 system in the differentiating culture. Low FOXO1A levels increased proliferative activity and tube formation of the developed hESC-EC. FOXO siRNA had no effect (gray panels) on differentiation and cell death profile. Color images available online at www.liebertpub.com/scd