Fig 1: mRNA expression profiling in bAVMs. A, Gene expression heatmap of differentially expressed mRNAs (P≤0.05 and fold change ≥2 or ≤0.5) in HFR vs LFR bAVM tissues. The x‐axis shows each bAVM patient (black=HFR; gray=LFR), and the y‐axis shows individual genes. In the heatmap cells, red indicates high gene expression (ie, upregulated expression) relative to the median expression; green indicates low expression (ie, downregulated expression); black indicates that expression is similar to the median. B, Top 15 terms of GO analysis (ranked by P value) and KEGG analysis enriched by upregulated genes in LFR bAVMs. C, The expression levels of FZD10, MYOC, and downstream molecules in canonical Wnt signaling. bAVM indicates brain arteriovenous malformations; HFR, high flow rate; LFR, low flow rate.
Fig 2: Effects of FZD10 and MYOC on HUVECs and HBVSMCs. A, Western blotting analysis of HUVECs and HBVSMCs transfected with a pcDNA3 vector overexpressing FZD10/MYOC or negative control (CTRL). B, Effects of FZD10/MYOC on migration of HUVECs and HBVSMCs. The scale bar corresponds to 100 μm. C, Effects of FZD10/MYOC on tube formation of HUVECs and HBVSMCs. The scale bar corresponds to 200 μm. One representative experiment out of 5 is shown. *P<0.05; **P<0.025; ***P<0.001. HBVSMCs indicates human brain vascular smooth muscle cells; HUVECs, human umbilical vein endothelial cells.
Fig 3: Wnt signaling is influenced by FZD10 or MYOC in HUVECs and HBVSMCs. Immunofluorescent staining for β‐catenin in HUVECs (A) and HBVSMCs (B) transfected with a pcDNA3 vector overexpressing FZD10/MYOC or negative control (CTRL). C, Nuclear β‐catenin level was quantified by measuring fluorescence intensity in the cell nucleus. Data were derived from 3 randomly selected fields. D, RT‐qPCR for AXIN2 and TCF‐1 expression in HUVECs and HBVSMCs transfected as in (A, B). E, Western blotting analysis for total JNK (t‐JNK), p‐JNK (Thr183/Tyr185), and β‐catenin in HUVECs and HBVSMCs transfected as in (A, B). *P<0.05; **P<0.025; ***P<0.001. The scale bar corresponds to 200 μm. These data are representative of 3 independent experiments. DAPI indicates 4′,6‐diamidino‐2‐phenylindole; HBVSMCs, human brain vascular smooth muscle cells; RT‐qPCR, reverse transcription quantitative polymerase chain reaction; HUVECs, human umbilical vein endothelial cells.
Fig 4: Detection of FZD10 and MYOC in bAVM tissue samples. Immunohistochemical staining of bAVM tissue samples with differential flow rate subtypes show strong staining for FZD10 (A) and MYOC (B) in LFR bAVM tissue. Endothelial cells lining the vascular lumen (white arrows) and vascular smooth muscle cells in the vessel wall (black arrows) both show staining for FZD10 and MYOC. The scale bar corresponds to 200 μm. C, Semiquantitative grading of FZD10 and MYOC expression levels in the vascular structure of bAVMs. ***P<0.001. bAVM indicates brain arteriovenous malformations; HFR, high flow rate; LFR, low flow rate.
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