CL-316243 facilitates stable atherosclerotic plaque phenotypes in association with suppression of perivascular adipose tissue ferroptosis via upregulating C/EBPβ

Figures

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  Fig. 1. Ferroptosis in established atherosclerotic PVAT. (A) Heatmap representing the genes differentially expressed between control PVAT and atherosclerotic PVAT, as determined by RNA-sequencing analysis, in which data were sorted as log (fold change) ≥ 1.5 or ≤ 1.5 and p < 0.05 (n = 5 per group). (B) Dot plot of GO items enriched in differentially expressed genes in control PVAT and atherosclerotic PVAT based on RNA-sequencing data. (C) Dot plot of KEGG items enriched in differentially expressed genes in control PVAT and atherosclerotic PVAT based on RNA-sequencing data. (D) mRNA levels of genes closely associated with ferroptosis in PVAT from control and atherosclerotic groups, n = 6. (E-F) TUNEL staining in PVAT from control and atherosclerotic groups. Representative images are shown in (E) with quantification of normalized TUNEL positive cells in (F). Scale bar represents 50 μm, n = 6. (G-H) Immunohistochemical analysis of 4-HNE in PVAT from control and atherosclerotic groups. Representative images are shown in (G) with quantification of normalized 4-HNE-positive area in (H). Scale bar represents 50 μm, n = 6. (I–K) Measurement of normalized Fe2+ content (I), MDA (J), and GSH/GSSG…

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  Fig. 2. CL suppresses ferroptosis in vitro and in vivo. (A) Cell viability of arterial PVAT-derived adipocytes after treatment with vehicle control (Ctrl), Ox-LDL (150 μg/mL), Ox-LDL (150 μg/mL) plus the ferroptosis inhibitor ferrostatin-1 (Fer-1, 5 μM), or Ox-LDL (150 μg/mL) plus CL (1 μM). Cells were first pretreated with PBS (as vehicle control) or Ox-LDL for 24 h, followed by a second 24-h treatment with Ox-LDL in combination with either vehicle (DMSO), Fer-1, or CL. n = 3. (B–C) Analysis of mitochondrial membrane potential in adipocytes under the treatment conditions described in (A), as assessed by flow cytometry. Representative histograms of JC-1 and FITC are shown in (B), and the normalized JC-1 and FITC median fluorescence intensity (MFI) is shown in (C) (n = 3). (D-E) Analysis of mitochondrial ROS in adipocytes under the treatment conditions described in (A), as assessed by flow cytometry. Representative histograms of MitoSOX and PE are shown in (D), and the normalized MitoSOX and PE MFI is shown in (E) (n = 3). (F) Representative transmission electron microscopy images of mitochondrial morphology in adipocytes treated as described in (A). Scale bar, 1 μm. (G-I) Determination…

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  Fig. 3. CL-316243 improves arterial PVAT function in atherosclerotic mice. (A) ELISA analysis of TNF-α, IL-1β and IL-6 levels in the conditioned medium of arterial PVAT from saline- or CL-treated mice. The quantification data are from at least 6 independent biological samples per group. (B) The relative mRNA level of vasoactive factors and genes involved in inflammation in arterial PVAT from saline- or CL-treated mice, n = 6. (C–F) Histological analysis of UCP1 in thoracic aortic PVAT (tPVAT) and abdominal aortic PVAT (aPVAT) from saline- or CL-treated Apoe−/− mice. Representative IHC images of UCP1 are shown in (C, E) with quantification of the average optical density (AOD) of UCP1 in tPVAT and aPVAT in (D, F). Scale bar, 50 μm. n = 6. (G-H) Western blotting analysis of UCP1 and PGC-1α in arterial PVAT of saline- or CL-treated mice. Representative blots are shown in (G) with normalized quantification in (H). (I-L) Cellular and mitochondrial energy metabolism in isolated arterial PVAT-derived adipocytes from saline or CL treated Apoe−/− mice. The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of adipocytes are shown in (I-J) and (K-L), respectively. (n = 6).…

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  Fig. 4. CL-316243 facilitates stable plaque phenotypes in mice with established atherosclerosis. (A-B) Histological analysis of atherosclerotic plaques in the whole aortas of saline or CL treated mice. Representative Oil red O-stained images are shown in (A) and quantification of the percentage of lesion area to surface area of whole aorta (n = 8) is shown in (B). (C-D) Histological analysis of atherosclerotic plaques in aortic roots from saline or CL treated mice. Representative images of Oil red O-stained aortic roots are shown in (C) with quantification of lesion area in (D) (n = 8). Scale bar, 200 μm. (E-F) Representative images of H&E and Movat staining of aortic roots from saline or CL treated mice. The necrotic cores are indicated by arrows. Scale bar, 200 μm at low magnification and 50 μm at high magnification. (G) Quantification of the percentage of necrotic core area to plaque area in aortic root according to Movat staining (n = 6). (H) Masson staining of aortic roots from saline or CL treated mice. Scale bar, 200 μm at low magnification and 50 μm at high magnification. (I) Quantification of the content of collagen in plaque (n = 6). (J) Quantification of the minimum fibrous…

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  Fig. 5. CL-316243 promotes expression of the ferroptosis suppressor GPX4 in arterial PVAT via cAMP-PKA-C/EBPβ axis. (A) Relative mRNA expression levels of Gpx4, Nrf2, Slc27a1, Atf4, Aifm2, and Tfr1 in arterial PVAT from saline- or CL-treated mice (n = 6). (B) Relative Gpx4 mRNA levels in arterial PVAT-derived adipocytes transfected with NC si or Cebpb si. (C-D) WB analysis of GPX4 protein expression in adipocytes transfected with NC si or Cebpb si. Representative blots are shown in (C), and the normalized quantification is shown in (D). (E-F) Enrichment and genome browser view of negative controls (IgG) and C/EBPβ at the GPX4 promoter in 3T3-L1 adipocytes. (G) Predicted sites of the putative C/EBPβ binding motif by the JASPAR database. (H) Luciferase activity in 293T cells transfected with GPX4-WT or five GPX4-Muts (GPX4-Mut1-5) promotor plasmid in the presence of vector or C/EBPβ plasmid for 48 h. The relevant luciferase activity values were based on the Renilla control (n = 6). (I-J) WB analysis of C/EBPβ protein expression in arterial PVAT from saline or CL treated Apoe−/− mice. Representative blots are shown in (I), and the normalized quantification is shown in (J) (n = 6). (K)…

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  Fig. 6. Cebpb deficiency abrogates the inhibitory effect of CL-316243 on Ox-LDL-induced adipose ferroptosis in arterial PVAT-derived adipocytes. (A) Normalized values of adipocyte viability as determined by CCK-8 assays (n = 6). Cells were transfected with NC si or Cebpb si followed by treatment with Ox-LDL (150 μg/mL) in the presence or absence of CL (1 μM). Forty-eight hours post-siRNA transfection, cells were first pretreated with PBS (as vehicle control) or Ox-LDL for 24 h, followed by a second 24-h treatment with Ox-LDL in combination with either vehicle (DMSO) or CL. n = 3. (B–C) TUNEL assay of arterial PVAT-derived adipocytes under the treatment conditions described in (A). Representative images are shown in (B), and the normalized quantification is shown in (C) (n = 6). Scale bar, 50 μm. (D-E) Analysis of mitochondrial membrane potential in adipocytes under the treatment conditions described in (A), as assessed by flow cytometry. Representative histograms of JC-1 and FITC are shown in (D), and the normalized JC-1 and FITC median fluorescence intensity (MFI) is shown in (E) (n = 3). (F-G) Analysis of mitochondrial ROS in adipocytes under the treatment conditions described in (A),…

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  Fig. 7. Model for the mechanism of CL-316243 in facilitating stable plaque phenotypes in established atherosclerosis. Upon Ox-LDL stimulation, lipid peroxide overload promotes arterial PVAT ferroptosis, thereby leading to arterial PVAT dysfunction, resulting in releasement of vasoactive factors to aggravate vascular inflammation. CL-316243 binds to β3-adrenoceptor and activates cAMP-PKA-CREB signaling pathway to increase C/EBPβ expression, thus transcriptionally promoting the expression of phospholipid hydroperoxidase GPX4, resulting in suppression of arterial PVAT ferroptosis and arterial PVAT dysfunction, consequently reducing vascular inflammation, thereby facilitating stable plaque phenotypes in established atherosclerosis.