Fig. 4: Fibroblast-specific inducible overexpression of EFF causes induction of TET, which is followed by transition of fibroblast to vasculogenic fibroblast in vivo. | Nature Communications

Fig. 4: Fibroblast-specific inducible overexpression of EFF causes induction of TET, which is followed by transition of fibroblast to vasculogenic fibroblast in vivo.

From: Vasculogenic skin reprogramming requires TET-mediated gene demethylation in fibroblasts for rescuing impaired perfusion in diabetes

Fig. 4: Fibroblast-specific inducible overexpression of EFF causes induction of TET, which is followed by transition of fibroblast to vasculogenic fibroblast in vivo.The alternative text for this image may have been generated using AI.

A pTARGATT-CAG-L4SL-Fli IRES-Etv2-T2A-Foxc2-PolyA was constructed for the generation of Foxc2, Etv2, and Fli1 knock-in (EFFfl/fl) mouse model. The EFFfl/fl mice were crossed with Col1a2CreER mice (EFFfl/flCol1a2CreER) to specifically induce EFF expression in Col1a2 expressing cells upon tamoxifen application. EFFfl/flCol1a2CreER mice were also crossed with Tet1/2/3fl/fl to generate EFFfl/flTet1/2/3fl/flCol1a2CreER mice. Additional mouse colony Tet1/2/3fl/fl Col1a2CreER was generated. Model characterization is provided in Figs S9, 10. B Schematic diagram of hind-limb experiment and vascular imaging in: (i) EFFfl/fl, (ii) EFFfl/flCol1a2CreER, (iii) EFFfl/flTet1/2/3fl/fl Col1a2CreER and (iv) Tet1/2/3fl/flCol1a2CreER. Tamoxifen treatment was done for 5 days post-surgery. Representative PeriMed laser speckle–assisted limb perfusion images (C) and quantification (D) of hind limb perfusion at different time points in abovementioned 4 groups (n = 8, 8, 4, 4). *EFFfl/flCOL1A2creER vs EFFfl/fl; §EFFfl/flCOL1A2creER vs EFFfl/flTet1/2/3fl/flCOL1A2creER; #EFFfl/flCOL1A2creER vs Tet1/2/3fl/flCOL1A2creER(one-way ANOVA, Tukey HSD post-hoc-test). E Immunofluorescence images of hind-limb ischemic skin (top) and colocalization quantification (bottom) of COL1A2 (green) and VWF (red) staining of the abovementioned groups (B) was determined by Pearson correlation coefficient (r). White arrow represents colocalization. Data expressed as mean ± S.D (n = 6, 7, 4, 4). (one-way ANOVA, Tukey HSD post-hoc-test). F Immunofluorescence image of day 14 hind limb ischemic tissue in EFFfl/flCOL1A2creER mice stained for COL1A2 and VWF. The left panel shows the 3D-reconstruction image of the colocalized COL1A2 and VWF rendered by IMARIS (inset). The middle panel shows the magnified inset. The right panel shows the colocalized area for COL1A2 and VWF. Scale, 30 µm. G Bisulfite sequencing analyses of endothelial specific gene {Cdh5 (left), Vwf (middle) and Pecam1 (right)} promoter methylation in LCM captured COL1A2+ elements for at d14 hind-limb ischemic skin of abovementioned (B) groups (methylated CpG, black; unmethylated CpG, white). Number of clones = 6. Venn diagram shows the distribution of methylated and unmethylated CpGs in promoter of each gene. AU, arbitrary unit. Source data are provided as a Source Data file. n = biological replicates in (D, E). A, B are created in BioRender. Mohanty, S. (2024) BioRender.com/e89c803.

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