Fenofibrate attenuates the adverse effects of radiation on endothelial cells through modulation of ROS-NO signalling and inflammation

Figures

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  Fig. 1. The effect of fenofibrate on HCAECs viability and apoptosis. The viability of HCAECs treated with fenofibrate (Feno) was measured using Presto Blue high sensitivity assay (PB-HS) (A) and Cell Titer Glo (CTG) assay (B) 2d and 7d after irradiation. The cell cycle distribution was measured by PI staining and flow cytometry (C). An example of the measurement for the 7d time point is shown for the 0 Gy DMSO (i), 0 Gy fenofibrate (ii), 4 Gy DMSO (iii), and 4 Gy fenofibrate (iv). The subG1 proportion of the cells was quantified as an assessment for apoptosis and normalized for the cell counts (per 106 cells) (D). The apoptotic markers were measured using qPCR, 2d (E) and 7d (F) after irradiation. The error bars represent the standard deviation (±SD). For the Presto Blue assay, multiple unpaired t-tests with a 1 % FDR, Two-stage step-up (Benjamini, Krieger, and Yekutieli) correction was conducted. For the CTG, qPCR and SubG1 analysis, a Two-way ANOVA, Tukey's multiple comparisons test was performed (∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001; n = 3).

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  Fig. 2. Changes in the PPARα phosphorylation in irradiated HCAECs with fenofibrate treatment. PPARα phosphorylation was measured using fluorescence microscopy and western blots. For microscopy, the nucleus was stained with DAPI, the phospho-Ser12 PPARα was visualized using the AF488 channel and the actin-filaments with the AF555 channel at the 2d (A and B) and 7d (C and D) after irradiation with and without fenofibrate (Feno). Images were acquired using identical exposure times and sensitivity settings for all treatments at each time point. Fluorescence signals from the AF488 channel were quantified and normalized to the number of cells per frame (B–D). Immunoblotting of phospho-Ser12 PPARα and quantification is shown in (E) and (F). The error bars represent the standard deviation (±SD). For microscopy, multiple unpaired t-tests with a 1 % FDR, Two-stage step-up (Benjamini, Krieger, and Yekutieli) correction was conducted. For immunoblotting, a Two-way ANOVA, with Tukey's multiple comparisons test was conducted (∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001; n = 3).

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  Fig. 3. Changes in the proteome of irradiated HCAECs with and without fenofibrate treatment. The 2D and 3D PCA were performed on normalized intensities of all proteins. (A–C). The shared proteins between the irradiated DMSO- and 10 μM fenofibrate (Feno) -treated samples were shown in the venn diagram (D–E). The most significant pathways altered in both proteome profiles, 2d (F) and 7d (G) after irradiation were shown. The pathways shown in the plot were ranked by significance in the proteome of irradiated DMSO-treated cells, with additional pathways unique to the fenofibrate-treated proteome included for comparison (Table 1, Table 2). Pathways were compared based on odds ratio and -log p-value, and referenced by numbers in Table 1, Table 2 The size of each bubble represents the number of proteins identified in the corresponding pathway. DMSO shows proteome of 4 Gy irradiated vs sham irradiated DMSO-treated cells. Feno shows proteome of 4 Gy irradiated vs sham irradiated fenofibrate-treated cells. The analyses were generated by Enrichr (https://maayanlab.cloud/Enrichr/).

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  Fig. 4. Analysis of the PI3K-AKT-eNOs pathway. qPCR and ELISA analysis of the components of PI3K-AKT-eNOS pathway were performed in HCAECs (A–F). The expression level of mRNA was measured using qPCR, 2d (A) and 7d (B) after irradiation with and without fenofibrate (Feno). The expression levels of AKT and p-AKT both serine and threonine and eNOS and p-eNOS proteins were compared using ELISA after 2d (C and E) and 7d (D and F). The levels of NO produced in the HCAECs were measured at the 2d and 7d after irradiation (G). The error bars represent the standard deviation (±SD) (Two-way ANOVA, Tukey's multiple comparisons test; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001; n = 3).

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  Fig. 5. Analysis of the oxidative stress response. Levels of intracellular ROS were measured using flow-cytometry in HCAECs, 2d and 7d after irradiation with and without fenofibrate (Feno). An example for the 2d sample set is shown in (A), and quantification for the replicates is shown in (B). Accumulated intracellular oxidative and nitrosative damage were assessed by measuring levels of MDA (C) and levels of 3-NT (D). qPCR analysis of key components of oxidative stress response were performed in HCAECs 2d (E) and 7d (F) after irradiation. Activities of NOX (G) and SOD (H) were measured. The error bars represent the standard deviation (±SD) (Two-way ANOVA, Tukey's multiple comparisons test; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001; n = 3).

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  Fig. 6. Analysis of the interferon signalling. qPCR analysis of the components of interferon pathway were performed in HCAECs, 2d (A) and 7d (B) after irradiation with and without fenofibrate (Feno). The immunoblotting of free ISG15 protein (C and D) and ISGylated proteins 2d and 7d after irradiation (E). The release of ISG 15 in the supernatant (SNT) after 2d and 7d was quantified using a slot-blot (F). The error bars represent the standard deviation (±SD) (Two-way ANOVA, Tukey's multiple comparisons test; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001; n = 3).

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  Fig. 7. Analysis of cytokines, interleukins and endothelial mesenchymal transition markers. qPCR analysis of inflammation markers 2d (A) and 7d (B), inflammatory regulators 2d (E) and 7d (F) and EndMT markers 2d (G) and 7d (H) were performed in irradiated HCAECs with and without fenofibrate (Feno). The release of different cytokines including GM-CSF, MCP-1 and IL-6 which were quantified using flow cytometry after 2d (C) and 7d (D). The error bars represent the standard deviation (±SD) (Two-way ANOVA, Tukey's multiple comparisons test; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001; n = 3).

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  Fig. 8. Proposed mechanism of action of fenofibrate in irradiated HCAECs in-vitro. Irradiation reduced NO signalling via inactivation of the PI3K–AKT–eNOS pathway, whereas fenofibrate reactivated this pathway and restored NO production (A). Consistent with this, irradiation increased ROS generation, NOX activity, MDA levels, and 3-NT levels, while fenofibrate mitigated this effect (B). Irradiation also triggered an inflammatory response, which was counteracted by fenofibrate (C). The released cytokines contribute to inflammation, changes in cytoskeleton organisation and initiation of EndMT, and fenofibrate effectively attenuated the processes (D). Alterations in the pathways described above play a crucial role in the remodelling of vascular endothelial cells involved in the initiation and progression of atherosclerosis. Fenofibrate acts to reduce or restore the effects of irradiation on these pathways (E–F). Solid lines indicate correlations validated in this study, while dashed lines indicate unvalidated correlations.