ETS transcription factor ETV2 directly converts human fibroblasts into functional endothelial cells

Abstract

Transplantation of endothelial cells (ECs) is a promising therapeutic approach for ischemic disorders. In addition, the generation of ECs has become increasingly important for providing vascular plexus to regenerated organs, such as the liver. Although many attempts have been made to generate ECs from pluripotent stem cells and nonvascular cells, the minimum number of transcription factors that specialize in directly inducing vascular ECs remains undefined. Here, by screening 18 transcription factors that are important for both endothelial and hematopoietic development, we demonstrate that ets variant 2 (ETV2) alone directly converts primary human adult skin fibroblasts into functional vascular endothelial cells (ETVECs). In coordination with endogenous FOXC2 in fibroblasts, transduced ETV2 elicits expression of multiple key endothelial development factors, including FLI1, ERG, and TAL1, and induces expression of endothelial functional molecules, including EGFL7 and von Willebrand factor. Consequently, ETVECs exhibits EC characteristics in vitro and forms mature functional vasculature in Matrigel plugs transplanted in NOD SCID mice. Furthermore, ETVECs significantly improve blood flow recovery in a hind limb ischemic model using BALB/c-nu mice. Our study indicates that the creation of ETVECs provides further understanding of human EC development induced by ETV2.

Keywords: ETV2; angiogenesis; direct conversion; endothelial cells.

Fig. 1.

Optimal ETV2 expression levels evoke endothelial properties from HAFs. HAFs at 14 d after ETV2 infection were analyzed. (A, B, F, and G) Flow cytometry analysis. The percentage of CD31⁺ cells in each population is shown in F and G. (C and H) Quantitative RT-PCR. Gene expression levels are relative to HPRT1. *P < 0.05; **P < 0.01, two-sided Student t test. (D) Whole-cell lysates subjected to Western blot analysis. ETV2 expression levels are relative to β-actin. (E) Dil-AcLDL uptake assay. Red, Dil-AcLDL; green, Venus. Data are representative of four independent cell cultures [mean ± SD; n = 4 cultures (B and G) or triplicate (C and H)]. Neg, negative; Lo, low; Inter, intermediate, Hi, high. (Scale bar: 50 μm.)

Fig. 2.

Higher ETV2 expression levels suppress ETVEC induction from HAFs. Dox-inducible ETV2 and rtTA-transduced HAFs were cultured in the presence of various Dox concentrations. (A) At 14 d after culture, Venus⁺ cells were subjected to quantitative RT-PCR. Gene expression levels are relative to HPRT1 (mean ± SD; triplicate). (B and C) At 14 d after the culture, EC induction was determined by flow cytometry analysis. Gated on 7-AAD⁻Venus⁺ cells. Absolute numbers of ETVECs are shown in C. Data are representative of three independent cell cultures.

Fig. 3.

Transient ETV2 expression is sufficient to directly convert part of HAFs into ETVECs. (A) Dox-inducible ETV2 and rtTA-transduced HAFs were cultured in the presence of 100 ng/mL Dox for 21 d. The sorted CD31⁺ cells were cultured for another 10 d in the presence or absence of Dox. CD31^hi cells were sorted again, then cultured for an additional 10 d under the same culture conditions. Numbers on the contour plots indicate the percentage of cells under a Dox-free culture condition. (B) At 20 d after the Dox withdrawal, CD31^hi and CD31⁻ cells were subjected to quantitative RT-PCR. Gene expression levels relative to HPRT1 (mean ± SD; triplicate). (C) Photos of the two representative EC colonies at 20 d after the culture with or without Dox. Data are representative of three independent cell cultures. Hi, CD31^hi cells; Neg, CD31⁻ cells. (Scale bar: 50 μm.)

Fig. 4.

Endogenous FOXC2 in HAFs is essential for ETVEC induction. (A) Expression levels of FOXC genes in HAFs. (B–D) ETV2 was transduced into FOXC2 shRNA- and control shRNA-expressing HAFs. (B–D) Fifteen days later, Venus⁺ cells were subjected to quantitative RT-PCR analysis (B) and flow cytometry analysis (C and D). (B) Blue and red bars indicate FOXC2 and control shRNA, respectively. Gene expression levels are relative to HPRT1 (mean ± SD; triplicate). *P < 0.01, two-sided Student t test. (C and D) Plots represented by gates on 7-AAD⁻Venus⁺ cells. Results reported as percentage of CD31⁺ cells in the Venus⁺ cells. (D) Blue and red histograms indicate FOXC2 and control shRNA, respectively. Data are representative of three independent cell cultures.

Fig. 5.

ETVECs represent proliferative ECs. (A) HAFs at 15 d after ETV2 infection were subjected to flow cytometry analysis. The dot plots are represented by gates on 7-AAD⁻Venus⁺ cells. The pink quadrangle indicates ETVECs. (B) Purifying ETVECs through sorting CD31⁺ cells. (C) Absolute numbers of ETVECs. Three representative experiments are presented. (D) Microscopic images of ETVECs (Exp.1) at 32 d after ETV2 transduction. (Left) Low-magnification image. (Right) High-magnification image. (E) Heat-map image and hierarchical clustering of the DNA microarray data. (F and G) Quantitative RT-PCR for transcription factors (F) and EC effector molecules (G) performed with HAFs (black), ETVECs (pink), and HUVECs (blue). Gene expression levels are relative to HPRT1. (H) Flow cytometry analysis of HAFs and ETVECs. Red and blue lines indicate targets and isotype controls, respectively. (I) Immunofluorescence cytostaining. (J) Dil-AcLDL uptake assay. (K) Capillary-like structure formation on Matrigel-coated plates. Data are representative of 10 independent cell cultures (B) or four independent cell cultures (A, C, and F–K). Data are mean ± SD. n = 10 cultures (B) or triplicate (F and G). (Scale bars: 50 μm in D, I, and J; 300 μm in K.)

Fig. 6.

ETVECs establish mature functional vasculature in vivo. (A–F) Images of Matrigel plugs extracted from NOD SCID mice 28 (A–D and F) and 42 (E) days after the implantation of ETVECs (A–F) and HAFs (A). (B and D) The fluorescence channels were merged over bright-field pictures. White arrows indicate that erythrocytes were circulating in ETVEC-constituting vasculature. (E) Three-dimensional structure of the ETVEC-constituting vasculature. (F) Immunofluorescence images showing human CD34 (hCD34) (Left), human/mouse eNOS (h/m eNOS) (Center), and a merged image (Right) (Hoechest 33342 in blue). (G–K) Hind limb ischemia model of BALB/c-nu mice at 14 d after transplantation. (G) Representative photographs. White arrows indicate the right ischemic hind limbs. (H) Hematoxylin and eosin staining of the adductor muscles of ischemic limbs. (I) Doppler images of superficial blood flow in lower limbs. Red to white color and dark-blue color on the image indicate high and low perfusion signals, respectively. White arrows indicate the right ischemic hind limbs. (J) Comparison of perfusion recovery in ischemic hind limbs. Data are mean ± SD (PBS, n = 10; HAFs, n = 10; ETVECs, n = 6). *P < 0.03; **P < 0.001, one-way ANOVA. (K) Immunofluorescence images of the cell-transplanted adductor muscles. Data are representative of three independent experiments. (Scale bars: 50 μm.)