Fig 2: CITED2 overexpression inhibits VSMC apoptosis and delays AAA progression in mice. VSMCs were transfected with KD-EPAS1 and OE-NC or OE-CITED2 (n = 3 independent experiments). (A) The mRNA expression of CITED2 in VSMC was assessed using RT-qPCR (n = 3 independent experiments). (B) Effect of overexpression of CITED2 on apoptosis in VSMC detected by flow cytometry. Mice were delivered with lentivirus-encapsulated overexpression CITED2 plasmid based on knockdown of EPAS1, followed by AAA modeling and Am gavage. (C) The mRNA expression of CITED2 in the aortic tissues of AAA mice was examined using RT-qPCR. (D) VSMC apoptosis in AAA mice was analyzed using TUNEL. Scale bar, 50 µm. (E) The protein expression of SM22 and α-SMA, VSMC contractile markers, in the aorta of AAA mice was assessed using Western blot analysis. (F) The aortic pathology in AAA mice was observed using HE staining. Scale bar, 50 µm. (G) The collagen deposition in the aorta of AAA mice was observed using Masson staining. Scale bar, 50 µm. (H) In vivo measurement of abdominal aortic diameter in mice by Doppler ultrasonography. (I) Ex vivo measurement of the maximum diameter of the abdominal aorta in mice. (J) IL-6, TNF-α, and IL-1β levels in aortic tissue homogenates were determined using ELISA. n = 6/group; *P < 0.05. Data were presented as mean ± SD and analyzed using unpaired t-tests or two-way ANOVA.

Fig 3: Schematic illustration. Am treatment promotes EPAS1 activation of CITED2 transcription, thereby inhibiting the pro-inflammatory response of macrophages and VSMC apoptosis to alleviate the development of AAA.

Fig 4: Ang II-induced decrease in EPAS1 expression in AAA mice. (A) Differentially expressed genes between AAA and normal mouse aortic tissues in the GSE197748 data set. (B) The intersection of differentially expressed genes in data set GSE197748 and prediction results of microbe–target and its metabolite–target from the gutMGene v1.0 data set. (C) Functional annotations for intersecting genes at DAVID Bioinformatics Resources. (D) PPI network of intersecting genes mainly involved in transcription regulation. (E) EPAS1, STAT1, STAT4, and EP300 mRNA expressions in the aortic tissues of AAA mice were assessed using RT-qPCR. (F) Metabolite propionate produced by Am promotes EPAS1 expression. n = 6/group; *P < 0.05. Data were presented as mean ± SD and were analyzed using a two-way ANOVA.

Fig 5: EPAS1 mediates transcriptional activation of CITED2. (A) Transcriptional targets predicted to be regulated by EPAS1 in mice in the TRRUST database. (B) The intersection of predicted transcriptional targets of EPAS1 and differentially expressed genes in the GSE197748 dataset. (C) Effect of Ang II, Am treatment, and knockdown of EPAS1 on CITED2 and RUNX2 mRNA expression in mouse abdominal aortic tissues detected by RT-qPCR. (D) Effects of Ang II treatment and knockdown of EPAS1 on CITED2 mRNA expression in VSMC detected by RT-qPCR (n = 3 independent experiments). (E) The binding of EPAS1 and CITED2 promoter was analyzed using ChIP (n = 3 independent experiments). (F) Transcriptional regulation of CITED2 by EPAS1 was analyzed using a dual-luciferase reporter assay (n = 3 independent experiments). n = 6/group; *P < 0.05. Data were presented as mean ± SD and analyzed using unpaired t-tests one-way, or two-way ANOVA.