Low-Dose Radiation Affects Cardiovascular Disease Risk in Human Aortic Endothelial Cells by Altering Gene Expression under Normal and Diabetic Conditions

Figures

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  Figure 1. Characterization of functions of primary human aortic endothelial cells (HAECs) and human aortic endothelial cells, diabetes type II (T2D-HAECs). (A) Representative images of cultured HAECs and T2D-HAECs. The proliferative rates of HAECs and T2D-HAECs were measured using an MTT assay (n = 4). (B) β-galactose staining and quantification in HAECs and T2D-HAECs. Blue represents β-galactose-positive staining (n = 3). (C) Images of tube formation in HAECs and T2D-HAECs 16 h after seeding of 1.5 × 104 cells/100 μL. The length and number of tube junctions were counted and compared in each group (n = 6). (D) Western blotting of endothelial cell function-related proteins (n = 3). Quantification of each protein is shown in the graph. All data represent the mean ± standard deviation (SD) of each independent experiment; * p < 0.05; ** p < 0.01; *** p < 0.005; ns = no significance.

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  Figure 2. Analysis of differentially expressed genes (DEGs) in T2D-HAECs and normal HAECs. (A) Heatmap analysis with hierarchical clustering of DEGs in T2D-HAECs compared to normal HAECs. (B) Venn diagram of the comparisons between HAECs and T2D-HAECs following treatment with or without low-dose radiation. (C) The 10 most significantly altered pathways associated with the identified DEGs, according to KEGG analysis (p < 0.05, FDR q-value < 0.25). (D) Top 10 significant GO terms of biological processes associated with the identified DEGs.

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  Figure 3. DEG analysis of low-dose radiation-related DEGs in normal HAECs. (A) Heatmap analysis with hierarchical clustering of DEGs in HAECs treated with and without low-dose radiation. (B) Significant KEGG functional pathways associated with the identified DEGs in HAECs treated with and without low-dose radiation (p < 0.05, FDR q-value < 0.25). (C) Significant GO terms of biological processes associated with the DEGs identified in HAECs treated with and without low-dose radiation (p < 0.05). (D) The protein–protein interaction (PPI) networks in HAECs treated with and without low-dose radiation were visualized using the STRING plug-in of the Cytoscape program (version 3.9.1). Node colors represent the expression. The gradual change in color from blue to red corresponds to the change in the expression level (from down-regulation to up-regulation) in low-dose radiation-treated HAECs versus non-treated HAECs.

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  Figure 4. DEG analysis of low-dose radiation-related DEGs in T2D-HAECs. (A) Heatmap analysis with hierarchical clustering of DEGs in T2D-HAECs treated with 100 mGy irradiation. (B) Significant KEGG functional pathways related to DEGs in T2D-HAECs treated with 100 mGy irradiation (p < 0.05, FDR q-value < 0.25). (C) Significant GO term of associated biological processes in DEGs in T2D-HAECs treated with 100 mGy irradiation (p < 0.05). (D) The PPI networks associated with low-dose radiation responsiveness in T2D-HAECs visualized using the STRING plug-in of the Cytoscape program (version 3.9.1). Node colors represent the expression. The gradual change in color from blue to red corresponds to the change in the expression level (from down-regulation to up-regulation) in low-dose radiation-treated HAECs versus non-treated HAECs.

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  Figure 5. Fifteen candidate genes showing significantly modulated expression following low-dose radiation in both HAECs and T2D-HAECs. (A) Significant GO terms of biological process associated with the identified DEGs. (B) PPI network analysis identified the gene hub involved in the interferon signaling pathway.

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  Figure 6. Quantitative PCR validation of common low-dose radiation-responsive genes expressed in HAECs and T2D-HAECs. Values are represented as mean ± SD (n = 6). (A) Related gene expression of normal HAECs by 100 mGy radiation, ** p < 0.01, *** p < 0.005 vs. non-irradiated HAECs by Student’s t-test was considered statistically significant. (B) Related gene expression of T2D-HAECs by 100 mGy radiation, ## p < 0.01, ### p < 0.005 vs. non-irradiated T2D-HAECs by Student’s t-test was considered statistically significant. ns = no significance.

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  Figure 7. Evaluation of cell proliferation rate, cellular senescence, and tube formation in HAECs and T2D-HAECs after 100 mGy or 2 Gy of ionizing radiation treatments. (A) Representative images of cultured HAECs and T2D-HAECs. The proliferative rate of HAECs and T2D-HAECs was measured using an MTT assay (n = 4). (B) β-galactose staining and quantification in HAECs and T2D-HAECs. Blue represents β-galactose-positive staining (n = 3). (C) Images of tube formation in HAECs and T2D-HAECs 16 h after seeding 1.5 × 104 cells/100 μL. The length and the number of junctions in the tubes were counted and compared in each group (n = 4). All data represent the mean ± SD of three or four independent experiments; * p < 0.05, ** p < 0.01 vs. non-irradiated HAECs. # p < 0.05, ## p < 0.01 vs. non-irradiated T2D-HAECs.