The Functional Interaction of EGFR with AT1R or TP in Primary Vascular Smooth Muscle Cells Triggers a Synergistic Regulation of Gene Expression

Figure 1. EGF and U46619 lead to gene expression regulation in aVSMC but not AngII. E: EGF, U: U46619, EU: EGF and U46619, A: AngII, EA: EGF and AngII, EAU: EGF and AngII and U46619. (a) Number of differentially expressed genes (DEG) after 24 and 48 h incubation (compared to the respective control groups). (b,c) Heatmaps showing the normalized expression (log scale) of genes identified as significantly regulated for at least one comparison (EGF or U46619 or EGF and U46619-treated vs. control group) in the analyses for 24 h (b) or 48 h (c) incubation. A total of 4921 and 984 genes were thus included for each time point. Each row represents a gene, and each column a sample. Expression levels were additionally row-wise centered (subtraction of the mean from each value) and scaled (division by the standard deviation). Rows were clustered based on Euclidean distance (complete method, calculated by pheatmap, version 1.0.12—https://cran.r-project.org/package=pheatmap (accessed on 19 May 2022)). Heatmaps comprising all conditions are available in Supplementary Figure S3.

Figure 2. EGFR and TP act, in part, synergistically on gene expression regulation after 24 h (next page). (a) The overlaps of the lists of DEG after 24 h incubation are presented in an UpSet plot. Each row corresponds to a set of DEG and each column to one segment of a hypothetical Venn diagram. A black or a grey dot indicates that this set is included or not in this intersection, respectively. The names (i1 to i28) and the number of comprised genes are indicated for each intersection. The interactions between EGF, AngII and U46619 are indicated below the UpSet plot. Asterisks (*) mark intersections comprising genes regulated in a synergistic manner. “Qualitative-stimulation” and “-inhibition” means that an additional stimulus leads to or blocks the regulation of a new set of genes, respectively, in comparison to a simpler incubation. On the other hand, “quantitative-stimulation” or “-inhibition” means that genes had significantly different (p < 0.05) expected and measured fold changes when comparing single and multiple incubations (see Methods and Supplementary Table S2). (b) Histograms and density curves for the log2-fold changes (calculated by edgeR) of the genes comprised in…

Figure 3. EGFR and TP-activation triggers a qualitative but no quantitative synergistic regulation after 48 h. (a) Overlap of the lists of DEG after 48 h incubation. (b) Histograms and density curves for the log2-fold changes (calculated by edgeR) of the genes comprised in the intersection of the Venn Diagram. (left graph: down-regulated genes, right graph: up-regulated genes). The dotted lines correspond to the mean. The Kolmogorov–Smirnov test was used to assess if the shifts in distribution were statistically significant.

Figure 4. EGFR and TP putative regulation of cell cycle, cell death and metabolism-related pathways are time-dependent. (a) Overlaps of all lists of DEG after 24 and 48 h incubation. Only intersections comprising at least 5 genes are displayed. The name (i1 to i26) and the number of comprised genes are indicated for each intersection. The intersections are divided into three subgroups corresponding to “early” (DEG after 24 h incubation), “late” (48 h) and “lasting-effects” (24 and 48 h). (b) Direct comparison of the lists of DEG after 24 and 48 h for the two conditions (U46619 and “EGF and U46619”) that led to gene expression regulation at both time points. (c) The genes comprised in each intersection of the UpSet plot (Figure 4a) and in the different parts of the Venn diagram (Figure 4b) were used as inputs for GO term enrichment analysis. The figure displays the main enriched terms and is based on the same principle as an UpSet plot, with a dot in each concerned row. The intersection numbers of the UpSet plot or miniature Venn diagram symbols indicate which lists of genes were used as the input.

Figure 5. Effect of EGFR and TP receptor activation on apoptosis, cell cycle and senescence. (a) Caspase-3 activity was measured after 24 and 48 h incubation with EGF and/or U46619. The control condition (media without stimuli) was used as a reference for each independent replicate (n = 5–6). (b) DNA synthesis rate was measured with BrdU incorporation after 24 h incubation with EGF and/or U46619. The supplemented media used for the usual cultivation of aVSMC (+) served as a positive control. The control condition (media without stimuli) was used as a reference for each independent replicate (n = 6). (c–e) β-gal activity was measured in the green channel (after staining with C12FDG) of a fluorescence microscope, while Hoechst staining (blue channel) permitted the identification of cell nuclei (blue object) (n = 6). (c) Because the green signal mostly co-localized with the blue one, the β-gal activity of a single cell was defined as the mean green intensity of the corresponding blue object. (d) In order to determine which objects showed an enhanced β-gal activity (senescent cells), the mean green intensity of blue objects was plotted for the control (lower signal) and U46619-treated…

Figure 6. EGFR and U46619 synergistically regulate two cardiovascular relevant genes, Spp1 and Klf15. (a) FPM for Spp1 and Klf15 genes. The dotted line corresponds to the 5 FPM threshold employed to filter out lowly expressed genes (* p < 0.05—t-test). (b) Relative mRNA expression of Spp1 and Klf15 measured by qPCR. Outliers were removed, and the control group was used as a reference for each independent replicate. (n = 8–9, * p < 0.05—Wilcoxon test). (c) The amount of secreted OPN after 48 h incubation was measured by ELISA and normalized to the total amount of protein isolated from the cells cultivated with the measured media. The U46619-treated group served as a reference as the values for the control groups were close to the background ones (n = 4, * p < 0.05—Wilcoxon test).