RhoA/ROCK Pathway Activation is Regulated by AT1 Receptor and Participates in Smooth Muscle Migration and Dedifferentiation via Promoting Actin Cytoskeleton Polymerization

Abstract

Background: In this study, we investigated the mechanism of Rho GTPases signaling on Ang II-mediated cell migration and dedifferentiation in human aortic vascular smooth muscle cells (HA-VSMCs) and an Ang II-infusion mouse model.

Methods: Cells were pretreated with different inhibitors or Ang II. Cell migration was detected by Wound healing and Transwell assay. Mice were treated with Ad-RhoA-shRNA virus or Irbesartan or fasudil and then infused with Ang II.

Results: Ang II treatment induced HA-VSMCs migration in a dose- and time-dependent manner and reduced the expression of VSMC contractile proteins. These effects were significantly suppressed by the inhibition of Ang II type 1 receptor (AT1 receptor), RhoA, and Rho-associated kinase (ROCK). Furthermore, Ang II treatment promoted the activation of RhoA and ROCK, which was reduced by AT1 receptor inhibition. Meanwhile, Ang II treatment induced F-actin polymerization, which was inhibited after ROCK inhibition. In mice, Ang II infusion increased VSMC migration into the neointima and reduced VSMC differentiation proteins levels, and these effects were shown to be dependent on AT1 receptor and RhoA/ROCK pathway.

Conclusion: This study reveals a novel mechanism by which Ang II regulates RhoA/ROCK signaling and actin polymerization via AT1 receptor and then affects VSMC dedifferentiation.

Keywords: RhoA/ROCK; actin; angiotensin II; dedifferentiation; migration.

Figure 1

The effects of Ang II on cell migration and the expression of contractile marker proteins in human aortic vascular smooth muscle cells (HA-VSMCs). Cells were treated with Ang II at different concentrations (0, 10, 100, 1000 nM) or different time points (0, 3, 6, 12, 24 h). Untreated cells were used as the control. (A and B) Cell migration was detected by wound-healing and Transwell assays after Ang II treatment. Histograms show the quantification of the wound healing and Transwell assay results. The scar bar is 50 μm. * p < 0.05 and ** p < 0.01 vs. the control group (n = 3). (C and D) Western blot analysis of MYH11, SM22α, and smoothelin. Histograms show the ratios of myosin heavy chain (MYH11) or SM22α or smoothelin to β-tubulin. * p < 0.05 and ** p < 0.01 vs. the control group; # p < 0.05 and ## p < 0.01 vs. the group treated with 10 nM Ang II; $ p < 0.05 vs. the group treated with 100 nM Ang II (n = 3).

Figure 2

The effects of Ang II receptor inhibitors on cell migration and the expression of contractile marker proteins in HA-VSMCs. Cells were pretreated with candesartan (5 μM) for 6 h or PD123319 (5 μM) for 6 h and then exposed to 100 nM Ang II for 24 h. Untreated cells were used as the control. (A) Cell migration was detected by wound-healing and Transwell assays. Histograms show the quantification of the wound healing and Transwell assay results. The scar bar is 50 μm. ** p < 0.01 vs. the control group; # p < 0.05 vs. the Ang II-treated group (n = 3). (B) Western blot analysis of MYH11, SM22α, and smoothelin. Histograms show the ratios of MYH11 or SM22α or smoothelin to β-tubulin. * p < 0.05 vs. the control group; # p < 0.05 vs. the Ang II-treated group (n = 3).

Figure 3

The effects of AT1 receptor inhibitor candesartan on the expression and activation of Rho GTPases and Rho-associated kinase (ROCK). Cells were pretreated with candesartan (5 μM) for 6 h and then exposed to 100 nM Ang II for 24 h. Untreated cells were used as the control. (A) Detection of Rho GTPases activity by pulldown assay. Histograms show the ratios of Rho GTPases GTP-bound to total Rho GTPases. * p < 0.05, vs. the control group; # p < 0.05 vs. the Ang II-treated group (n = 3). (B) Western blot analysis of the expression of ROCK1, ROCK2, MYPT1, and phospho-MYPT1 (Thr696). Histograms show the ratios of ROCK1 or ROCK2 to β-tubulin or phospho-MYPT1 to total MYPT1. * p < 0.05, vs. the control group; # p < 0.05 vs. the Ang II-treated group (n = 3).

Figure 4

The effects of RhoA inhibitor CCG-1423 on cell migration and the expression of contractile marker proteins in HA-VSMCs. Cells were pretreated with CCG-1423 (20 μM) for 18 h and then exposed to 100 nM Ang II for 24 h. Untreated cells were used as the control. (A) Cell migration was detected by wound-healing and Transwell assays. Histograms show the quantification of the wound healing and Transwell assay results. The scar bar is 50 μm. ** p < 0.01 vs. the control group; # p < 0.05 vs. the Ang II-treated group (n = 3). (B) Western blot analysis of MYH11, SM22α, and smoothelin. Histograms show the ratios of MYH11 or SM22α or smoothelin to β-tubulin. * p < 0.05 and ** p < 0.01 vs. the control group; # p < 0.05 and ## p < 0.01 vs. the Ang II-treated group (n = 3).

Figure 5

The effects of Rac1 inhibitor NSC23766 on cell migration and the expression of contractile marker proteins in HA-VSMCs. Cells were pretreated with NSC23766 (50 μM) for 30 min and then exposed to 100 nM Ang II for 24 h. Untreated cells were used as the control. (A) Cell migration was detected by wound-healing and Transwell assays. Histograms show the quantification of the wound healing and Transwell assay results. The scar bar is 50 μm. ** p < 0.01 vs. the control group (n = 3). (B) Western blot analysis of MYH11, SM22α, and smoothelin. Histograms show the ratios of MYH11 or SM22α or smoothelin to β-tubulin. * p < 0.05 vs. the control group (n = 3).

Figure 6

The effects of Cdc42 inhibitor ZCL278 on cell migration and the expression of contractile marker proteins in HA-VSMCs. Cells were pretreated with ZCL278 (50 μM) for 30 min and then exposed to 100 nM Ang II for 24 h. Untreated cells were used as the control. (A) Cell migration was detected by wound-healing and Transwell assays. Histograms show the quantification of the wound healing and Transwell assay results. The scar bar is 50 μm. ** p < 0.01 vs. the control group (n = 3). (B) Western blot analysis of MYH11, SM22α, and smoothelin. Histograms show the ratios of MYH11 or SM22α or smoothelin to β-tubulin. * p < 0.05 vs. the control group (n = 3).

Figure 7

The effects of ROCK inhibitor Y27632 on cell migration and the expression of contractile marker proteins in HA-VSMCs. Cells were pretreated with Y27632 (20 μM) for 30 min and then exposed to 100 nM Ang II for 24 h. Untreated cells were used as the control. (A) Cell migration was detected by wound-healing and Transwell assays. Histograms show the quantification of the wound healing and Transwell assay results. The scar bar is 50 μm. ** p < 0.01 vs. the control group; # p < 0.05 and ## p < 0.01 vs. the Ang II-treated group (n = 3). (B) Western blot analysis of MYH11, SM22α, and smoothelin. Histograms show the ratios of MYH11 or SM22α or smoothelin to β-tubulin. * p < 0.05 vs. the control group; # p < 0.05 vs. the Ang II-treated group (n = 3).

Figure 8

The effects of Y27632 pretreatment on F-actin polymerization in Ang II-treated HA-VSMCs. Cells were pretreated with Y27632 (20 μM) for 30 min and then exposed to 100 nM Ang II for 24 h. Untreated cells were used as the control. (A) The ratio of F-actin to G-actin analyzed by G-actin/F-actin In Vivo Assay Kit and Western blot analysis. Histogram shows the ratio of F-actin to G-actin. * p < 0.05 vs. the control group; # p < 0.05 vs. the Ang II-treated group (n = 3). (B) Phalloidin conjugates for staining F-actin filaments. Immunofluorescence assay for F-actin (red) and nuclei stained with DAPI (blue). The scar bar is 20 μm.

Figure 9

The effects of RhoA/ROCK pathway suppression or AT1 receptor inhibition on VSMC migration in Ang II-infused mice. Ang II was subcutaneously infused at a rate of 1000 ng/kg/min using the Alzet Model 1002 osmotic minipump. The group infused with normal saline was used as the control. The right common carotid arteries of mice were injected and incubated with 1 ×10 ¹⁰ pfu virus solution specific for RhoA knockdown for 20 min. The AT1 receptor inhibitor Irbesartan (50 mg/kg/d) and ROCK inhibitor fasudil (30 mg/kg/d) were added to drinking water for consumption by mice. (A) Masson’s trichrome staining of carotid arteries 14 days after Ang II infusion. Masson’s trichrome staining shows the collagen fibers (blue) and muscle fibers (red). We marked the location of the intima, media, and adventitia. Histogram shows the ratio of muscle fiber area in the intima. The scar bar is 50 μm. ** p < 0.01 vs. the saline-infused group; ## p < 0.01 vs. the model group (Ang II infusion + Ad-GFP virus) (n = 10). (B) Immunofluorescence assay for α-SMA (green) and CD31 (red) as double staining (merge). Nuclei were stained with DAPI (blue). White arrows display that Ang II promotes the expression of α-SMA in the neointima, while α-SMA expression remains in the media in the control and other groups. The scar bar is 20 μm.

Figure 10

Immunofluorescence staining of MYH11, SM22α, and smoothelin (green). Nuclei were stained with DAPI (blue). Mice were treated the same way as in tablee 7. Histograms show the fluorescence intensity of the staining in the intima. * p < 0.05 vs. the saline-infused group; # p < 0.05 vs. the model group (Ang II infusion + Ad-GFP virus) (n = 10). The scar bar is 50 μm.