GV1001, hTERT Peptide Fragment, Prevents Doxorubicin-Induced Endothelial-to-Mesenchymal Transition in Human Endothelial Cells and Atherosclerosis in Mice

Figures

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  Figure 1. Effect of GV1001 on doxorubicin-induced EndMT and migration of HUVECs. (A) Representative immunofluorescent-staining images showing CD31 (red), α-SMA (green), and DAPI (blue) in HUVECs after a 48h exposure to doxorubicin (0.25 μM) and/or GV1001 (10 μg/mL). (B,C) display the quantification of the fluorescent intensity of CD31 and α-SMA per represented field, analyzed with ImageJ. Statistical significance is indicated as follows: ns (not significantly different), ** p < 0.01, **** p < 0.0001 with n = 5 per group. Scale bar: 20 μm. (D) Representative images of migrated HUVECs exposed to GV1001 (10 μg/mL), TNF-α (100 ng/mL), doxorubicin (0.25 μM), or TNF-α+ GV1001 or doxorubicin + GV1001, as assessed with the transwell chamber assay. Images are shown at 40× magnifications. (E) Quantification of the number of migrated cells per field. Statistical analysis was performed using one-way ANOVA: ns (not significantly different), * p < 0.05, *** p < 0.001, **** p < 0.0001 with n = 5 per group.

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  Figure 2. GV1001 reverses the overexpression of proinflammatory cytokines induced by doxorubicin in HUVECs. (A) Reversal effect of GV1001 on doxorubicin-induced upregulation of TGF-β1, TGF-β2, Smad3, and Smad4 in HUVECs. Statistical analysis was conducted using one-way ANOVA. (B) Reversal effect of GV1001 on doxorubicin-induced upregulation of IL-1α, IL-1β, and IL-6 expression in HUVECs. Statistical analysis was conducted using the one-way ANOVA. Significance levels are indicated as follows: ns = not significantly different, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Results are presented as mean ± SEM. All experiments were performed in triplicate.

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  Figure 3. (A) Representative immunofluorescent staining images of NF-κB p65 (green) in HUVECs after 48 h exposure of cells to doxorubicin (0.25 μM), GV1001 (10 ug/mL), or doxorubicin + GV1001 (10 μg/mL). (B) Percentage of the cells with p65 in the nuclei per field using ImageJ analysis. ns: not significantly different, **** p < 0.001. Scale bar: 20 μm. (C) Western blotting of phosphorylated p65 (p-p65) and p65 in cells exposed to vehicle only, GV1001 (2 or 10 μg/mL), doxorubicin (0.25 μM), or in a combination of GV1001 with doxorubicin. All experiments were performed in triplicate.

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  Figure 4. (A,C) Representative immunofluorescent staining images showing mitochondrial ROS (red) and cytoplasmic ROS (blue) in cells after 48 h exposure to doxorubicin (0.25 μM), GV1001 (10 μg/mL), or a combination of doxorubicin and GV1001 (10 μg/mL). (B,D) ROS staining intensity was quantified using ImageJ Software. (E) Relative cellular ATP levels. Statistical significance: ns = not significant, * p < 0.05, ** p < 0.01, **** p < 0.0001. Scale bar: 20 μm. All experiments were conducted in triplicate.

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  Figure 5. (A) Representative immunofluorescent-staining images of mitochondria (red) in fixed HUVECs after 48 h exposure to doxorubicin (0.25 μM), GV1001 (10 μg/mL), or a combination of doxorubicin and GV1001 (10 μg/mL). (B) Mitochondrial length measured using ImageJ analysis. Statistical significance: ns, not significant; *** p < 0.001; and **** p < 0.0001. Scale bar: 20 μm. (C,E) Representative immunofluorescent staining images of mitochondrial ferrous iron (Mito-Fe2+), peroxidated lipid (MitoPeDPP staining), and nucleus (stained with Hoechst nucleic acid staining: Hoechst 33342) of non-fixed HUVECs. (D,F) The levels of ferrous iron and peroxidated lipid were measured using ImageJ analysis. Statistical significance: ns, not significant; *** p < 0.001; and **** p < 0.0001. Scale bar: 10 μm. All experiments were performed in triplicate.

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  Figure 6. (A) Reversing effect of GV1001 on doxorubicin-induced increases in serum levels of proinflammatory cytokines in ApoE-deficient mice. Cytokine levels were measured using an ELISA, as described in the Methods section. Statistical analysis was conducted using one-way ANOVA. Symbols denote significance levels: ns, not significantly different; * p < 0.05; ** p < 0.01, **** p < 0.0001. (B) Inhibitory effect of GV1001 on doxorubicin-induced increases in the expression of TNF-α, IL-1β, and IL-6 in the arterial tissue. Gene expression was measured via RT-qPCR, with Gapdh serving as a loading control. Statistical analysis was conducted using one-way ANOVA, with significance indicated as follows: ns = not significantly different, * p < 0.05, ** p < 0.01, **** p < 0.0001. Results are presented as means ± SEM, based on 7-8 samples.

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  Figure 7. GV1001 inhibited the doxorubicin-induced arterial plaque formation: Inhibition of lipid deposition and macrophage/monocyte infiltration into the arterial wall. (A) Representative photographs of mouse arteries from the en face preparation after staining with Oil Red O (8–10 mice per group). (B) Quantification of areas of artery stained by Oil Red O using ImageJ analysis. ns, not significantly different; ** p < 0.01, *** p < 0.001. (C) Representative Oil Red O staining of aortic root from the mice. Red color: Lipid; L: Lumen; LF: Leaflet; P: Plaque; (D) Representative images of immunofluorescent staining with MOMA-2 (red) in the aortic root of the mice. Nuclei were stained with DAPI (blue). L: Lumen; AW: Arterial Wall; LF: Leaflet; M: Macrophages/Monocytes. Scale bar: 100 μm.