Physiological cyclic stretch up-regulates angiotensin-converting enzyme 2 expression to reduce proliferation and migration of vascular smooth muscle cells

Figures

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  Figure 1. Physiological stretch regulates ACE2 expression and activity, as well as other components of RAS in HASMCs HASMCs grown on six-well Flexcell plates (105/well) coated with collagen I were kept under static conditions or subjected to 10% stretch 1, 3, 6, 12, 24 h. (A) The mRNA levels of ACE2, ACE, MAS were determined by RT-PCR. (B) The ACE2 protein expression was determined by Western blot. (C) The enzymatic activity of ACE2 was evaluated by SELDI-TOF-MS method. (D and E) The levels of Ang-(1-7) and Ang II were determined by ELISA. Data are shown in mean ± SEM from at least three independent experiments; *P<0.05, **P<0.01 vs. static control (0 h).

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  Figure 2. ACE2 is involved in regulating proliferation and migration of HASMCs mediated by stretch Cultured HASMCs with different conditions were exposed to 10% stretch for 12 h; cells kept under static conditions were considered as control group. (A–C) The proliferation of HASMCs in each group was determined by BrdU incorporation assay and cell counting method, respectively. (D and E) The cell migration was evaluated by a scratch test. Data are shown in means ± SEM from at least three independent experiments. **P<0.01 vs. static control; #P<0.05, ##P<0.01, vs. stretch alone.

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  Figure 3. The effect of stretch on ACE2 promoter activity and mRNA stability (A) Cultured HASMCs were co-transfected with pGL3-ACE2 promotor-luci and phRL-TK vectors for 24 h, then exposed to 10% stretch or kept under static conditions for 3 h. The ACE2 promoter activity was determined by dual-luciferase assay and the fold changes in ACE2 promoter activity were calculated in comparison with the static control. (B) Cultured HASMCs were pretreated with transcription inhibitor actinomycin D (5 μg/ml) for 12 h, then cells were exposed to 10% stretch or kept under static conditions for 0, 6, 12 h; the ACE2 mRNA expression was determined by RT-PCR. Data are shown as means ± SEM from three independent experiments. *P<0.05 compared with static control.

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  Figure 4. AP-1 and NF-κB were implicated in the regulation of ACE2 mediated by stretch (A) Potential binding sites of transcription factors in the promoter region of human ACE2 gene by TRANSFAC analysis (marked with gray boxes). (B) Cultured HASMCs were subject to 10% stretch for 0, 15, 30, 60, 120 min. Protein lysates were collected to detect the levels of c-jun, c-fos, p65, Sp-1, and their phosphorylated forms by Western blot. (C and D) Cultured HASMCs were incubated with inhibitors of NF-κB (SN50,18 μM), Sp-1(WP631, 100 nM), AP-1 (Curcumin, 10 μM) for 1 h, then were exposed to 10% stretch for 6 h. RT-PCR and Western blot were performed to detect ACE2 mRNA and protein expression. (E and F) Cultured HASMCs were exposed to 10% stretch for 0, 1, 3, 6 h. The nuclear protein was extracted and the DNA binding activities of AP-1 and NF-κB were determined by EMSA. (G and H) HASMCs were exposed to 10% stretch or kept under static conditions for 1 h, then cells were fixed, the expression and location of c-jun and c-fos were investigated by immunofluorescence. DAPI was used to visualize cell nuclei. Data are means ± SEM from at least three independent experiments. *P<0.05, **P<0.01 vs. static…

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  Figure 5. PKCβII and JNK1/2 signaling pathways were involved in stretch-induced ACE2 expression (A) Effect of stretch on activations of mitogen-activated protein kinase (MAPK), Akt, and PKCβII pathways. Cultured HASMCs were subjected to 10% stretch for 0, 15, 30, 60, 180 min. The phosphorylation of ERK1/2, p38, JNK1/2, Akt and PKCβII were determined by Western blot. (B and C) Cultured HASMCs were pretreated with PKCβII inhibitor (CG53353, 100 nM), Akt inhibitor (LY294002, 50 μM), p38 inhibitor (SB203580, 10 μM), JNK1/2 inhibitor (SP600125, 20 μM), ERK1/2 inhibitor (PD98059, 30 μM) for 1 h respectively. DMSO was used as a negative control, then were exposed to 10% stretch for 6 h. RT-PCR and Western blot were performed to detect ACE2 mRNA and protein expression. (D and E) The effect of blockade of PKCβII and JNK1/2 pathways on AP-1 and NF-κB DNA binding activities in stretched cells. HASMCs were incubated with AP-1 inhibitor (Curcumin, 10 μM), NF-κB inhibitor (SN50, 18 μM), PKCβII inhibitor (CG53353, 100 nM), and JNK1/2 inhibitor (SP600125, 20 μM) for 1 h, respectively, then exposed to 10% stretch for 3 h. Cells kept under static conditions were considered as static control cells. The…

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  Figure 6. Scheme of the roles of ACE2 in physiological stretch mediated cellular functions of HASMCs and the underlying molecular mechanism