Mechanical Stretch Induces Smooth Muscle Cell Dysfunction by Regulating ACE2 via P38/ATF3 and Post-transcriptional Regulation by miR-421

Figures

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  FIGURE 1. Elevated mechanical stretch suppresses ACE2 and increases ACE expression in the aorta in vivo. (A) ACE2 and ACE mRNA expression in the aortas of the aortic constriction group and the sham group by RT-qPCR. (B,C) ACE2 and ACE protein levels in the aortas of the aortic constriction group and the sham group by Western blot. (D) Representative immunohistochemical staining of ACE2 and ACE in sections from the aortas of the aortic constriction group and the sham operation group. (E) Representative photomicrographs of immunohistochemical staining of ACE2 in the paracancerous tissues of patients with hypertension or without. Values are expressed as means ± SEM. *P < 0.05 vs. sham (n = 4).

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  FIGURE 2. Mechanical stretch modulates ACE2, ACE, Ang-(1–7), and Ang II expression in vascular smooth muscle cells (VSMCs) in vitro. (A) Schematic diagrams of the mechanical stretch loading system in the current study. VSMCs were subjected to 18% 1 Hz mechanical stretch for the indicated time. (B,E) RT-qPCR analysis of ACE2 and ACE mRNA in VSMCs under mechanical stretch for the indicated time. (C,F) Western blot analysis of ACE2 and ACE protein expression in VSMCs under mechanical stretch for the indicated time. (D,G) Levels of AngII and Ang-(1-7) in VSMCs after exposure to mechanical stretch were assayed by ELISA. Values are expressed as means ± SEM. *P < 0.05 vs. static (n = 4).

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  FIGURE 3. ACE2 was involved in mechanical stretch-induced vascular smooth muscle cells (VSMC) dysfunction. VSMCs were transfected with Ad-ACE2 or Ad-GFP for 24 h and then exposed to mechanical stretch (18% elongation, 1 Hz) for 12 h or not. (A,B) Representative views of BrdU-positive cells in different conditions and the percentage of BrdU-positive cells out of total cells. (C,D) Representative views of migration distance in different conditions. (E,F) Summarized data showing cell apoptosis as determined by flow cytometry analysis in VSMCs. (G,H) Western blot analysis of collagen I and collagen III in VSMCs with or without overexpression of ACE2. Values are expressed as means ± SEM. ∗P < 0.05 vs. Ad-GFP; #P < 0.05 vs. 18% stretch + Ad-GFP (n = 4).

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  FIGURE 4. p38 MAPK/ATF3 is involved in ACE2 expression in vascular smooth muscle cells (VSMCs) under 18% mechanical stretch. (A) Western blot analysis of the phosphorylation of MAPKs under 18% mechanical stretch for 0–12 h. *P < 0.05 vs. static group. (B) Western blot analysis of ACE2 in VSMCs under mechanical stretch pretreated with SB203580, SP600125, or PD98059. *P < 0.05 vs. static control. #P < 0.05 vs. 18% stretch control. (C) Western blot analysis of ACE2 in VSMCs under 18% stretch or not after transfecting with negative control or ATF3 siRNA. *P < 0.05 vs. NC group, #P < 0.05 vs. 18% stretch with NC group. (D) Western blot analysis of ATF3 in VSMCs under 18% mechanical stretch with or without pretreatment of SB203580. (E) Immunofluorescence using anti-ATF3 was performed under stretch or not.

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  FIGURE 5. ACE2 is a direct transcriptional target of ATF3. (A) The sequence logo of a potential ATF3 binding site in JASPAR. (B) The relative luciferase activities of the full ACE2 promoter and the truncated constructs in HEK-293T cells. *P < 0.05 vs. pGL3.10-Basic vector. (C) The relative luciferase activities of the full ACE2 promoter P in vascular smooth muscle cells under mechanical stretch for 6 h or not. *P < 0.05 vs. static control. (D,E) The relative luciferase activities of the full ACE2 promoter P co-transfected with pcDNA-ATF3 (or ATF3 siRNA). *P < 0.05 vs. control. (F) ChIP assay showing ATF3 directly bound to the ACE2 promoter (P6, at the -1,296 to -1,073 bp region). IgG, negative control; H3, positive control; ATF3, ATF3 antibody. (G,H) Luciferase activity analysis of P-P7 promoter with or without co-transfection of ATF3. #P < 0.05 vs. the corresponding promoter without co-transfection of ATF3 (n = 4).

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  FIGURE 6. miR-421 is involved in the expression of ACE2 under mechanical stretch. (A,B) RT-qPCR and Western blot analysis of ACE2 mRNA and protein expression in vascular smooth muscle cells (VSMCs) transfected with Dicer siRNA. (C) Venn diagram of the number of transcription factors in three libraries. (D) RT-qPCR analysis of the levels of miR-421 under 18% mechanical stretch for the indicated time. (E) Sequence alignment between miR-421 and putative binding sites in the 3′-UTR of ACE2 mRNA. (H) Effects of the miR-421 mimics on the activities of the luciferase reporter plasmids with the full-length (Wild) and mutated (Mut) ACE2-3′-UTR. HEK-293T cells were transfected with the ACE2-3′-UTR-WT and ACE2-3′-UTR-mut, together with miR-421 mimics, for 48 h. (F,G,I,J) RT-qPCR and Western blot analysis of ACE2 mRNA and protein expression in VSMCs transfected with miR-421 mimics (or inhibitor). (K) Western blot analysis of ACE2 protein expression in VSMCs under stretch for 12 h transfected with miR-421 inhibitor. Values are expressed as means ± SEM. *P < 0.05 vs. NC group (n = 4).