Fig 2: GPX4 expression positively correlated with the VSMC-specific phenotype in SHR and hypertensive patients. Immunofluorescent staining of CXCL2 (A) and AKR1C2 (B) in the arterial media of SHR. Immunofluorescence of CXCL2 (C) and AKR1C2 (D) on paraffin-embedded human normotension (NTN) and hypertension (HTN) internal mammary arteries. The GPX4 expression was detected by immunofluorescent staining in SHR (E) and human hypertensive patients (F). Nuclei were counterstained with Hoechst. Scale bar = 25 µM.

Fig 3: Ferroptosis inhibitor Fer-1 mitigates the HHP-induced VSMC phenotype switch. HASMCs in a 100 mmHg or 200 mmHg incubator were treated with or without 10 µM Fer-1 for 24 h. The GPX4 protein level was evaluated by immunofluorescent staining; the upper panel is the representative image, and the lower panel is the statistical graph (A). ROS production was determined by the ROS staining kit; the upper panel is the representative image, and the lower panel is the statistical graph (B). Mitochondrial ROS was visualized by use of the fluorescent probe MitoSOX Red (C). BODIPY 581/591 C11 lipid oxidation in HASMCs was measured by immunofluorescent staining; the statistical graph is the oxidized (orange signal) BODIPY 581/591 C11 fluorescence intensity (D). Confocal images of the CXCL2 expression (E) and AKR1C2 expression (F) in HASMCs. Nuclei were counterstained with Hoechst. All scale bar = 50 µM *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: ALKBH5 ablation promotes m6A RNA modification on Cxcl2 and Ifng mRNAs, decreasing their stability and expression.(A) m6A RIP-seq results from our published database (accession number GSE100048) (8) were obtained from CD4+ T cells isolated from WT mice, and peaks on Cxcl2, Ifng, and Cxcl10 mRNAs were then visualized using IGV. bp, base pair. (B) m6A RIP-qPCR assay was applied to detect m6A enrichment on Cxcl2, Cxcl10, and Ifng mRNA in CD4+ T cells isolated from the CNS of Alkbh5flox/floxCd4Cre mice and WT littermates after 14 days of EAE. Results were presented as fold increase versus immunoglobulin G and analyzed by unpaired t test. Actb, m6A negative control; Myc peak, m6A-positive control. (C) Degradation of Cxcl2 mRNA was detected in CD4+ T cells isolated from the CNS of Alkbh5flox/floxCd4Cre mice and WT littermates at 14 days of EAE after treating with actinomycin D for 0, 2, and 4 hours (n = 3); the values of residual RNAs were normalized to 0 hours. (D) Degradation of Ifng mRNA was detected in CD4+ T cells isolated from the CNS of Alkbh5flox/floxCd4Cre mice and WT littermates at 14 days of EAE after treating with actinomycin D for 0, 2, and 4 hours (n = 3); the values of residual RNAs were normalized to 0 hours. (E) Representative dot plots show the expression of CXCL2 on CD4+ T cells in the CNS of Alkbh5flox/floxCd4Cre mice and WT littermates after 14 days of EAE. FITC, fluorescein isothiocyanate. (F) Percentages of CXCL2-expressing CD4+ T cells in the CNS of Alkbh5flox/floxCd4Cre mice and WT littermates after 14 days of EAE (n = 5 to 7); results were analyzed by unpaired t test. (G) Working model summarize how ALKBH5 and m6A RNA modification regulate CD4+ T cell pathogenicity and promote autoimmunity during EAE. Data represent one of three independent experiments and are shown as the means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 5: Ferroptosis inducer RLS3 aggravates VSMC dysfunction in response to HHP. HASMCs were treated with RLS3 0.1 µM for 6 h in the presence of 200 mmHg. Immunofluorescent staining of GPX4 (red) in HASMCs (A). Intracellular ROS were measured by ROS staining; the upper panel is the representative image, and the lower panel is the statistical graph (B). HASMCs were labeled with the MitoSOX Red probe to detect mitochondrial ROS (C). Confocal images of the BODIPY 588/591 C11 staining; lipid oxidation was measured by the relative intensity of oxidized (orange fluorescence signal) BODIPY 581/591 C11 (D). CXCL2 (E), and AKR1C2 (F) protein levels were detected by immunofluorescent staining. All scale bar = 50 µM ***p < 0.001.