Early-phase impact of obesity-associated stress on murine vascular smooth muscle cells depends on EGFR and sex

Figures

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  Fig. 1. Differential gene expression analysis. Number of differentially expressed genes (DEG) after exposure to metabolic stressors (high glucose + free fatty acids, HG + FFA; humoral stressors (angiotensin II + noradrenaline; AII + NA) or the combination of both (ALL) in male wildtype VSMC (WT VSMC, A), EGFR-KO VSMC (KO VSMC, B) or endothelial cells (EC, C). For VSMC and each condition 4 independent biological replicates were included in the differential expression analysis, for EC 10 samples were included ( = total of 72 samples). The left panels show volcano plots for false discovery rates (FDR) and effect size (log2FC) determined either by edgeR or DESeq2 for cells exposed to all stressors. The tables to the right show the number of up (↑) or down (↓) regulated DEG after filtering for the log2FC and FDR thresholds. All FDR-, log2FC- and FPM-values for all three cell types are presented in Supplementary Data 16 and 17. D Three-dimensional mesh plot, generated by Sigma plot 14, combining the log2FC data for all protein coding RNA and lncRNA of WT VSMC for all three stressor conditions. The first plot shows the measured log2FC values for ALL stressors, the second plot the additive…

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  Fig. 2. Pathway and function enrichment analysis by IPA. A Analysis scheme. The log2FC-weighted lists of DEG served as input for the analysis to predict involved canonical pathways, upstream regulators and biofunctions as well as the direction and extend of change (z-score; activation or inhibition). B IPA results for canonical pathways using DEG of WT VSMC induced by exposure to all stressors. C The results for the canonical pathways predicted to be regulated by all stressors in at least one cell type were aligned. Presented pathways were filtered for z-score > 2 or < −2 and B-H corrected p value < 0.05. The complete data sets are shown in Supplementary Data 02–05. The genes used for the different enrichment analyses are presented in Supplementary Data 18–23.

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  Fig. 3. Diseases and biofunctions analysis. IPA results for diseases and biofunctions using DEG induced by exposure to all stressors. Presented functions were filtered for z-score > 2 or < −2 and B-H corrected p value < 0.05. Tables to the right show details of the analysis results for WT VSMC. The complete data sets are shown in Supplementary Data 06–09.

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  Fig. 4. Upstream regulator analysis. IPA results for upstream regulators using DEG induced by exposure to all stressors. Presented functions were filtered for z-score > 2 or < −2 and B-H corrected p value < 0.05. Tables to the right show details of the analysis results for WT VSMC. The complete data sets are shown in Supplementary Data 10–13.

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  Fig. 5. Cell cycle analysis. A Representative example of the nuclear DNA content ( = integral of DAPI fluorescence) distribution. B Baseline parameters and responsiveness to 10% FCS of WT VSMC and KO VSMC. N = 6 plates with 6 wells for each condition and each cell type. C Baseline parameters and responsiveness to 10% FCS of EC. N = 5 plates with at least 3 wells for each condition. Error bars above and below the box indicate the 90th and 10th percentiles.

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  Fig. 6. Analysis of DNA synthesis. Effect of the stressors on nuclear BrdU-incorporation ( = DNA synthesis) in WT VSMC, EGFR-KO VSMC and EC. In addition, the effect of EGF and of its combination with metabolic stressors on BrdU-incorporation were determined in WT VSMC and KO VSMC. Finally, the impact of the pharmaceutical EGFR-blockade (with AG1478) on the effect of all stressors was measured. N = 6 plates with 5 wells for each condition and each cell type. Error bars represent 95% confidence intervals.

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  Fig. 7. Effect of the stressors on cellular accumulation of neutral lipids. A Results of the stressors impact analysis for cellular lipid content in WT VSMC, KO VSMC and EC. B Exemplary microscopy images of VSMC and EC stained with LipidTox under control conditions and after exposure to all stressors. C Results of the stressors impact analysis for subcellular lipid content in WT VSMC, KO VSMC and EC. Spot-like lipid accumulation in the cell periphery (yellow arrow) and perinuclear accumulation were analyzed separately. N = 6 plates with 6 wells for each condition and each cell type. D Increased cellular lipid content resulted from triglyceride accumulation. N = 6. Error bars represent 95% confidence intervals.

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  Fig. 8. Analysis of mitochondrial function. A Baseline mitochondrial function in WT VSMC, KO VSMC and EC determined from the oxygen consumption rate (OCR). B–D Effect of the stressors on mitochondrial function. BR = basal respiration rate, PL = proton leak, ATP-OCR = ATP-synthesis dependent OCR, maxOCR = maximum OCR. N = 8 plates with up to 6 wells for each condition and each cell type. Error bars for scatter plots represent 95% confidence intervals. Error bars above and below the box indicate the 90th and 10th percentiles.

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  Fig. 9. Ca2+-induced cell contractility. A Representative example VSMC circularity distribution under control conditions and after acute addition of ionomycin. B Increase in median VSMC circularity after acute addition of ionomycin. Results from 10 independent experiments. C WT VSMC circularity after exposure to the stressors before and after acute addition of ionomycin. Ca2+-induced contractility was estimated from the ionomycin-induced change in circularity relative to the baseline circularity. N = 10 plates with 6 wells for each condition. D Direct comparison of the cellular circularities before and immediately after addition of ionomycin from cells exposed to control conditions or metabolic stressors. E KO VSMC circularity after exposure to the stressors before and after acute addition of ionomycin. N = 8 plates with 6 wells for each condition. F Modeling changes in cellular circular due acute cell shortening ( = contraction) assuming an ellipsoid shape. In this case a 20% shortening leads to a change in circularity from 0.26 to 0.37, similar to the values observed in VSMC. Error bars represent 95% confidence intervals.

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  Fig. 10. Comparing male and female cells. Effect of the stressors on (A) lipid accumulation in female WT VSMC and EC (N = 10 and 7 plates with 6 wells for each condition and each cell type), (B) DNA-synthesis in female WT VSMC and EC (N = 13 and 8 plates with 6 wells for each condition and each cell type) and (C) circularity (N = 9–10 plates with 6 wells for each condition) in female WT VSMC. Compare with the results from male VSMC in Figs. 6, 7 and 9. Error bars represent 95% confidence intervals.