Endothelial angiogenesis is directed by RUNX1T1-regulated VEGFA, BMP4 and TGF-β2 expression

<div><p>Tissue angiogenesis is intimately regulated during embryogenesis and postnatal development. Defected angiogenesis contributes to aberrant development and is the main complication associated with ischemia-related diseases. We previously identified the increased expression of RUNX1T1 in umbilical cord blood-derived endothelial colony-forming cells (ECFCs) by gene expression microarray. However, the biological relevance of RUNX1T1 in endothelial lineage is not defined clearly. Here, we demonstrate RUNX1T1 regulates the survival, motility and tube forming capability of ECFCs and EA.hy926 endothelial cells by loss-and gain-of function assays, respectively. Second, embryonic vasculatures and quantity of bone marrow-derived angiogenic progenitors are found to be reduced in the established <i>Runx1t1</i> heterozygous knockout mice. Finally, a central RUNX1T1-regulated signature is uncovered and VEGFA, BMP4 as well as TGF-β2 are demonstrated to mediate RUNX1T1-orchested angiogenic activities. Taken together, our results reveal that RUNX1T1 serves as a common angiogenic driver for vaculogenesis and functionality of endothelial lineage cells. Therefore, the discovery and application of pharmaceutical activators for RUNX1T1 will improve therapeutic efficacy toward ischemia by promoting neovascularization.</p></div

RUNX1T1 is sufficient to enhance angiogenic activity of endothelial cells.

<p>(A) Western blots showing for RUNX1T1 expression levels in 926-EC. VEC, vector control cells; ROV, ectopically <i>RUNX1T1</i>-expressing cells. The band intensity was quantified and normalized to control cells. (B) A histogram showing the relative viability of 926-EC measured by MTT assay. **, <i>p</i><0.01 (Student’s t test). Data represent mean ± S.D. n = 3 independent experiments. (C) A histogram showing the relative motility of 926-EC. ***, <i>p</i><0.001 (Student’s t test). (D) Representative images of transwell migration assays. Scale bar = 50 μm. (E) A histogram showing relative capillary formation ability of 926-EC. ***, <i>p</i><0.001 (Student’s t test). (F) Representative images for the tube formation assays. Scale bar = 50 μm. (G) A histogram showing the relative capillary branch number of 926-ECs. (H) A histogram for showing the relative capillary formation capacity of the ECFCs (left panel) and the HUVECs (right panel) of different passage numbers. P4, the fourth passaged cells; P6, the sixth passaged cells. *, <i>p</i><0.05; **, <i>p</i><0.01 (Student’s t test). (I) A histogram for showing the relative capillary branch number of the ECFCs (left panel) and the HUVECs (right panel) of different passage numbers. *, <i>p</i><0.05; **, <i>p</i><0.01 (Student’s t test). (J) RT-qPCR results for showing the relative expression levels of <i>RUNX1T1</i> in ECFCs (left panel) and HUVECs (right panel) of different passage numbers. ***, <i>p</i><0.001 (Student’s t test).</p

The decreased quantity of bone marrow-derived angiogenic progenitor cells and impaired aorta are observed in <i>Runx1t1</i> deficient mice.

<p>(A) A schematic representation of the gene-targeting strategy. The P1, P2 and P3 primers were used for the genotyping. The <i>loxP</i> sequence and a FRT-floxed neomycin cassette was introduced into intron 2 and another <i>loxP</i> sequence was inserted into intron 4 to allow Cre-ceconbinase-mediated removal of exons 2 and exon 3. E, exon; Kb, kilobase; Cre, cre-recombinase. (B) A Western blot showing the expression of Runx1t1 in the aorta of 6-month-old wild-type (+/+) and heterozygous <i>Runx1t1</i> knockout (+/-) mice. The band intensity was quantified and normalized to control cells. (C) The histograms for showing percentage of bone marrow-derived CD34 (+)/KDR (+) angiogenic precursor cells of indicated mice. *, <i>p</i><0.05 (Student’s t-test); **, <i>p</i><0.01 (Student’s t-test). Data represent mean ± S.D. n = 4 for each group (D) The quantification of thickness for aorta wall at indicated mice. n = 4 for each group, 6-month-old mice.*, <i>p</i><0.05 (Student’s t test). (E) The histological images of aortas from indicated mice by hematoxylin and eosin staining. n = 4 for each group. Scale bar = 100 μm. (F). Representative pictures for Evans blue extravasation assay in the 6-month-old wild-type (+/+) and heterozygous <i>Runx1t1</i> knockout (+/-) mice. Arrows indicated relative location of extravasation observed in heterozygous <i>Runx1t1</i> knockout (+/-) mice. Star indicated the location of liver. (G) The histogram for showing percentage of aberrant vessel permeability in <i>Runx1t1</i> deficient mice. n (total mice number used for analysis) = 9 and 8 for 6-month-old wild-type mice (+/+) and <i>Runx1t1</i> heterozygous mice (+/-), respectively. *, <i>p</i><0.05, (Fisher’s exact test). (H) The histogram for showing quantification of Evans blue extravasation in liver (upper panel) and heart (lower panel) of <i>Runx1t1</i> deficient mice (+/-).*, <i>p</i><0.05; **, <i>p</i><0.01 (Student’s t-test).</p

RUNX1T1 is required for angiogenic activity of ECFCs and HUVECs.

<p>(A) Representative pictures for illustrating the tube formation capacity of indicated cells. Scale bar = 50 μm (B) A histogram for showing endothelial tube lengths formed from the tube formation assay. N.D, none detected. Data represent mean± S.D. n = 3 independent experiments. (C) The RT-qPCR results for showing the relative expression levels of <i>RUNX1T1</i> at indicated cells. ***, <i>p</i><0.001 (one-way ANOVA). (D) A Western blot showing the expression level of RUNX1T1. The band intensity was quantified and normalized to control cells. Scr, scramble control cells; shT1, <i>RUNX1T1-</i>knockdowned cells. (E) A histogram for showing the relative viability of ECFCs measured by MTT assay. **, <i>p</i><0.01 (Student’s t test). (F) Representative images of the migration assay (upper panel) and the tube formation assay (lower panel). Scale bar = 50 μm. (G) A histogram for showing the relative motility of ECFCs. *, <i>p</i><0.05 (Student’s t test). (H) A histogram showing the relative capillary formation ability of ECFCs. *, <i>p</i><0.05 (Student’s t test). (I) A histogram for showing endothelial tube branch number from the tube formation assay. ***, <i>p</i><0.001 (Student’s t test). (J) RT-qPCR results for showing the relative expression levels of <i>RUNX1T1</i> in HUVECs. **, <i>p</i><0.001 (Student’s t test). (K) A histogram for showing the relative viability of HUVECs measured by MTT assay. *, <i>p</i><0.05 (Student’s t test). (L). A histogram for showing the relative motility of HUVECs. **, <i>p</i><0.01 (Student’s t test). (M). A histogram showing the relative capillary formation ability of HUVECs. **, <i>p</i><0.01 (Student’s t test). (N). A histogram showing the relative capillary branch number of HUVECs. **, <i>p</i><0.01 (Student’s t test). (O). The GSEA result showing a correlation between the core RUNX1T1 signature and the KSHV-infection signature (GSE16354). ES: enrichment score, FDR: false discovery rate.</p

RUNX1T1 regulates vaculogenesis of ECFCs <i>in vivo</i>.

<p>(A) Representative images for <i>in vivo</i> Matrigel plug assay. n = 3. Scale bar = 3 mm (upper panel) and 1mm (lower panel). Arrows indicated vessels formed. (B) The relative amounts of hemoglobin extracted from the excised matrigel plugs. *, <i>p</i><0.05 (one-way ANOVA) (C) A histogram showing the relative capillary formation ability in vivo matrigel plugs. *, <i>p</i><0.05 (one-way ANOVA), ***, <i>p</i><0.001 (Student’s t test). (D) A histogram showing the relative capillary branch number in vivo matrigel plugs. *, <i>p</i><0.05 (one-way ANOVA), **, <i>p</i><0.01 (Student’s t test).</p

VEGFA, BMP4 and TGF-β2 mediate RUNX1T1-directed growth, motility and capillary-forming capacity in endothelial cells.

<p>(A) The connectivity network of RUNX1T1-regulated genes created by Ingenuity Pathway Analysis. Yellow, secreted or membranic proteins; Green, cytosolic proteins; Red, nuclear proteins. (B-C) RT-qPCR results for validating expression levels of selected genes marked in yellow in panel A in 926-ECs (B) and <i>Runx1t1</i> deficient mice (C) **, <i>p</i><0.01; ***, <i>p</i><0.001 (Student’s t test). Data represent mean ± S.D. n = 3 independent experiments. (D) Western blotting for showing the expression levels of VEGF-A, TGF-β2 and BMP4 at the indicated models. The band intensity was quantified and normalized to corresponding control cells. (E) The immunoreactivity of VEGFA of heart vessel. n = 2 for each group, 6-month-old mice. Scale bar = 50 μm. (F) The immunoreactivity of BMP4 of heart vessel. n = 2 for each group, 6-month-old mice. Scale bar = 50 μm. (G) The immunoreactivity of TGF-β2 of heart vessel. n = 2 for each group, 6-month-old mice. Scale bar = 50 μm. (H) A histogram for showing the relative viability at the indicated conditions (VEC-CM, the conditional medium from 926ECs-vector control cells; ROV-CM, the conditional medium from RUNX1T1-expressing 926ECs; nAb, neutralizing antibody). *, <i>p</i><0.05 and **, <i>p</i><0.01 (one-way ANOVA). (I) A histogram for showing the relative capillary formation ability at indicated conditions. N.D, none detected. **, <i>p</i><0.01, (one-way ANOVA). (J) A histogram for showing the relative cell migration ability at indicated conditions. *, <i>p</i><0.05 and **, <i>p</i><0.01, (one-way ANOVA).</p