Research Paper Volume 13, Issue 8 pp 11610—11628

circACTA2 mediates Ang II-induced VSMC senescence by modulation of the interaction of ILF3 with CDK4 mRNA

circACTA2 is upregulated in Ang II-induced VSMCs and in the vascular walls of old mice. (A) SA-β-gal activity in VSMCs treated with or without Ang II (100 nmol/L) for 3 days. The percentage of SA-β-gal positive cells (above) and representative pictures (below) are shown. Magnification ×400. *P B) Western blot detection of p21, p16 and CDK4 expression in VSMCs treated with or without Ang II (100 nmol/L) for 3 days. (C) VSMCs were serum-starved for 24 h and then treated with or without Ang II (100 nmol/L) for 1, 3 and 5 days. circACTA2 expression was measured by qRT-PCR analysis. Data represent the means ± SEM of 3 independent experiments. **P ***P D) qRT-PCR detection of circACTA2 expression in the renal artery of hypertensive patients and matched controls. *P E) In situ hybridization of circACTA2 (red) in the renal artery of hypertensive patients and controls. (F) Western blot detected p21 and p16 expression in artery tissues of normal controls (N) and hypertensive patients (H). (G) qRT-PCR detected p21 and p16 mRNA expression in artery tissues of hypertensive patients and normal controls. **P I) SA-β-gal activity in VSMCs transfected with empty vector or circACTA2. The percentage of SA-β-gal positive cells (above) and representative pictures (below) are shown. Magnification × 400. *P J) SA-β-gal activity in VSMCs transfected with shCtrl or shcircACTA2 followed by treatment with or without Ang II. The percentage of SA-β-gal positive cells (left) and representative pictures (right) are shown. Magnification × 400. *P #P K) Western blot detection of p21 and CDK4 expression in VSMCs transfected with shCtrl or shcircACTA2 followed by treatment with or without Ang II.

Figure 1. circACTA2 is upregulated in Ang II-induced VSMCs and in the vascular walls of old mice. (A) SA-β-gal activity in VSMCs treated with or without Ang II (100 nmol/L) for 3 days. The percentage of SA-β-gal positive cells (above) and representative pictures (below) are shown. Magnification ×400. *P < 0.05 vs. vehicle control. (B) Western blot detection of p21, p16 and CDK4 expression in VSMCs treated with or without Ang II (100 nmol/L) for 3 days. (C) VSMCs were serum-starved for 24 h and then treated with or without Ang II (100 nmol/L) for 1, 3 and 5 days. circACTA2 expression was measured by qRT-PCR analysis. Data represent the means ± SEM of 3 independent experiments. **P < 0.01, ***P < 0.001 vs. vehicle control. (D) qRT-PCR detection of circACTA2 expression in the renal artery of hypertensive patients and matched controls. *P < 0.05 vs. control. (E) In situ hybridization of circACTA2 (red) in the renal artery of hypertensive patients and controls. (F) Western blot detected p21 and p16 expression in artery tissues of normal controls (N) and hypertensive patients (H). (G) qRT-PCR detected p21 and p16 mRNA expression in artery tissues of hypertensive patients and normal controls. **P < 0.01 vs. control. (H) Western blot detection of p21 and p16 expression in VSMCs transfected with empty vector or circACTA2. (I) SA-β-gal activity in VSMCs transfected with empty vector or circACTA2. The percentage of SA-β-gal positive cells (above) and representative pictures (below) are shown. Magnification × 400. *P < 0.05 vs. empty vector. (J) SA-β-gal activity in VSMCs transfected with shCtrl or shcircACTA2 followed by treatment with or without Ang II. The percentage of SA-β-gal positive cells (left) and representative pictures (right) are shown. Magnification × 400. *P < 0.05, #P < 0.05 vs. their corresponding control. (K) Western blot detection of p21 and CDK4 expression in VSMCs transfected with shCtrl or shcircACTA2 followed by treatment with or without Ang II.