Fig 1: Effects of LMO2, TAL1 silencing on endothelial cell migration and their downstream targets’ expression. Knockdown of components of the LMO2 complex was achieved using siRNA with cultured HUVEC. (A) Number of invading HUVECs transfected with siRNA per field is indicated in the figure. Cell counting was done in four randomly selected fields after 2 separate DNA transfections. Invasion of LMO2, TAL1 knockdown HUVECs into a Matrigel extracellular matrix was significantly (**P < 0.01) reduced compared with the transfection control (si-eGFP). (B) Cultured HUVECs were transfected with the indicated siRNA. Angiopoietin 2 (ANG-2) secretion into the culture medium after 24 h incubation was measured by ELISA. Statistically significant (*P < 0.05) decreases in ANG-2 secretion were observed in culture media obtained from si-LMO2-, si-TAL1-, and si-GATA2-transfected HUVECs, but not in that from si-LYL1-transfected HUVECs. (C,D) Cultured HUVECs were transfected with siRNA of LMO2, TAL1, LYL1, GATA2, and the transfection control si-eGFP. Relative mRNA expression levels of VEGFR1, VEGFR2, VEGFR3 (C), and NRP1, NRP2 (D) compared with those of si-eGFP transfection controls are shown. They were measured by real-time qPCR after normalization using GAPDH as an internal reference. Statistically significant (*P < 0.05) decreases are indicated.
Fig 2: Knockdown of LMO2, TAL1, and GATA2 leads to inhibition of endothelial sprouting and tube formation in vitro, but LYL1 knockdown does not. Knockdown of components of the LMO2 complex was achieved using siRNA with HUVEC spheroids. (A) Representative images from 3D spheroid-based in vitro angiogenesis assays and a statistical summary of CSL measurements from spheroids. Cultured HUVECs were transfected with siRNAs of eGFP, LMO2, TAL1, LYL1, and GATA2 as indicated in the figure. siRNA of eGFP was used as a transfection control. Relative mRNA levels (average of 2 separate transfections) of HUVECs transfected with each siRNA compared with that of si-eGFP-transfected cells are indicated below the names of the RNAi target genes below the statistical summary of CSL measurements. Note that sprouts with continuous tubes from the spheroids are counted in this analysis. Statistically significant (**P < 0.01) decreases in CSL were observed in spheroids transfected with siRNAs for LMO2, TAL1, and GATA2. (B) Cultured HUVECs were transfected with siRNAs of eGFP, LMO2 combined with TAL1, and LMO2 combined with GATA2 as indicated in the figure. siRNA of eGFP was used as a transfection control. Statistically significant (**P < 0.01) decreases in CSL were observed in spheroids transfected with LMO2/TAL1 and LMO2/GATA2 siRNAs (combined transfection). Combined knockdowns of LMO2/TAL1 and LMO2/GATA2 almost abolished sprouting from spheroids. Bar 200 μm.
Fig 3: Hypothetical mechanism of transcriptional regulation of hierarchical vascular tree formation. (A) In sprouting angiogenesis, activated endothelial cells upregulate TAL1 to form the transcription factor complex consisting of LMO2 and TAL1 in the nucleus, which upregulates angiopoietin 2, VEGFR2, and DLL4. After detachment of mural cells in response to angiopoietin 2, these cells then preferentially occupy the tip position in vessel sprouts (indicated by red cells). DLL4 on tip cells binds to Notch on adjacent cells to downregulate VEGFR2 (indicated by the dashed line). In contrast, endothelial cells in which LMO2 chiefly binds to LYL1 in the nucleus to transcriptionally upregulate VEGFR1 tend to occupy the stalk position (indicated by blue cells). LYL1 overexpression in tip cells downregulates DLL4 expression and upregulates VEGFR1, which results in hyperbranching of capillaries and slowing of tip cell migration at the periphery of the vascular tree. Only LMO2 partners, TAL1 and LYL1, are described in this figure. (B) The oligomeric transcription factor complex composed of E2A, TAL1 or LYL1, LMO2, LDB1 or LDB2, and GATA2 which regulates the expression of a number of downstream angiogenesis-specific genes.
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