Fig. 2: Endothelial Foxf2 deficiency causes BBB leakage and attenuates Tie2 signaling.
From: The stroke risk gene Foxf2 maintains brain endothelial cell function via Tie2 signaling
a, Experimental outline. Mice with EC-specific inactivation of Foxf2 (Foxf2iECKO) at 3 months were assessed for BBB integrity and BEC proteome. iv, intravenous. Panel a was created with BioRender.com. b, Confocal microscopy images of tracer extravasation (EB, 65 kDa; TMR-conjugated dextran, 40 kDa; CB-dextran, 10 kDa; A555-cadaverine, 1 kDa) in Foxf2iECKO versus Ctrl mice. Arrowheads indicate tracer extravasation (top) and cellular uptake (bottom). Scale bars, 2 mm and 0.5 mm (top), and 20 μm (bottom). HC, hippocampus. c, Quantification of tracer extravasation using fluorometry. The fluorescence intensities of all tracers were normalized to the autofluorescence signal of A488. d, Quantification of vessel density and fibrinogen (FIB) extravasation in histopathological sections from SVD patients and Ctrls (comparison by two-tailed unpaired t test, **P < 0.01; n = 6 patients per group; scale bar, 20 μm). e, Confocal microscopy images of Alb extravasation at the level of capillaries and arterioles, along with cellular uptake (scale bar, 10 μm). f, Whole-brain mapping of EB leakage of Foxf2iECKO versus Ctrl mice. Brain regions with significant EB leakage are highlighted in red (left). LSM images depict the distance-dependent intensity of EB along the brain vasculature, categorized into three concentric shells (right). g, Volcano plot of log2 LFQ ratios (Foxf2iECKO versus Ctrl) and −log10(P) of all quantified proteins from 6-month-old mice. Red and blue circles indicate proteins that were significantly upregulated and downregulated, respectively. Proteins marked with their corresponding gene names are associated with significantly enriched GO terms. h, Summary of LC–MS/MS and LFQ results. i, Subcellular localization of significantly dysregulated proteins. j, Enrichment analysis of biological processes of significantly dysregulated proteins in Foxf2iECKO versus Ctrl mice based on the GO terms (FE; count, number of significantly altered proteins; FDR, adjusted P value of significantly enriched terms, P < 0.05). k, FC and iBAQ intensity ranking of significantly altered proteins in Foxf2iECKO versus Ctrl mice. Red and blue lines indicate significantly upregulated and downregulated proteins, respectively, that are related to the Tie2-signaling pathway. l,m, Abundance of significantly downregulated proteins according to top-enriched Tie2-regulated biological processes (l, top, and m), and of significantly upregulated proteins related to ROS metabolic process and cellular response to oxidative stress (l, bottom). Comparison by two-tailed unpaired t test, P < 0.05 (c–g, l and m). Data are presented as mean ± s.d., ***P < 0.001; **P < 0.01; *P < 0.05 (c, d and m). n = 3 Ctrl-, EB and Dxt40, n = 3 iECKO-, Dxt10 and Cad1, n = 4 iECKO mice per group (c). n = 4 mice per group (e). n = 6 mice per group (f, l and m). The exact P values are presented in source data file. FC, fold change; iBAQ, intensity-based absolute quantification.
