Fig. 1

AGEs induce angiogenesis accompanying with pro-angiogenic genes upregulation. A Schematic diagram of the experimental design for the Matrigel plug assay. Matrigel containing HUVECs with or without AGEs (100 μg/mL) was injected subcutaneously into mouse. 7 days later, plugs were harvested and fixed with paraffin, sectioned, and stained with fluorescently labeled Ulex europaeus agglutinin I (UEA-I, Green) which binds HUVECs. B HUVECs-formed blood vessel in Matrigel plugs were measured by staining with UEA-I. Representative fluorescent images are shown. n = 4, scale bar indicates 100 μm. C Schematic diagram of the experimental design for the Aortic ring assay. Aorta dissected from mouse was cut into rings and then embedded in type I collagen and treated with or without AGEs (100 μg/mL). 6 days later, the rings were stained with isolectin B4 (IB4; green). D The mean number of sprout (indicated by red dot) from aortic ring was counted. n = 5, scale bar indicates 100 μm. E CCK8 assay was applied to evaluate the proliferation of HUVECs treated with AGEs (100 μg/mL) for 24 h. n = 5. F Transwell migration assay was conducted to evaluate the migration capacity of HUVEC treated with AGEs (100 μg/mL) for 24 h. n = 4, scale bar indicates 100 μm. G HUVECs were treated with AGEs (100 μg/mL) for 24 h and then embedded into Matrigel. The tube length and branching points were quantified using Image J. n = 4, scale bar indicates 200 μm. H Hierarchical clustering displays differential expression profiles of HUVECs treated with AGEs for 0, 2, 12 or 24 h. Columns represent individual samples and rows represent genes. Red and green indicate relatively high and low levels of gene expression, respectively, according to the saturation scale of color. n = 3. I Volcano plots show differentially expressed genes (DEGs) in the AGE-treated group for three different time points compared with the 0 h group. The blue points represent genes without significant changes, while the red and green points represent upregulated and downregulated genes respectively. n = 3. Threshold: DESeq2 P value < 0.05 |log2FoldChange|> 0. J Biological process terms from GO enrichment analysis of up-regulated genes in AGE-treated group compared with the Ctrl group. K Heatmap of upregulated genes enriched in the positive regulation of sprout angiogenesis. L qPCR assay was conducted to verify the mRNA expression of PLK2, PTGS2, JAK1, SMAD1, HDAC9, KDR, NRP1 in Fig. 1K. n = 9 to11. Data are shown as Mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001