F-actin/G-actin Ratio Assay Cells were seeded at 150 mm dish and processed with Y27632 or Ang II treatment

F-actin/G-actin Ratio Assay Cells were seeded at 150 mm dish and processed with Y27632 or Ang II treatment. 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. < 0.05 and ** < 0.01 vs. the control group (= 3). (C and D) Western blot analysis of MYH11, SM22, and smoothelin. Histograms show the ratios of myosin heavy chain (MYH11) or SM22 or smoothelin to -tubulin. * < 0.05 and ** < 0.01 vs. the control group; # < 0.05 and ## < 0.01 vs. the group treated with 10 nM Ang II; $ < 0.05 vs. the group treated with 100 nM Ang II (= 3). To evaluate the role of AT1 and AT2 receptors on Ang II-mediated cell migration and dedifferentiation, we examined the effects of AT1 or AT2 receptor inhibitor on HA-VSMCs migration and the expressions of MYH11, SM22, and smoothelin after 100 nM Ang II treatment for 24 h. As shown in Physique 2A, pretreatment with the AT1 receptor inhibitor candesartan obviously reduced Ang II-mediated cell migration in HA-VSMCs. In contrast, pretreatment with the AT2 receptor inhibitor PD123319 did not significantly affect HA-VSMCs migration at 24 h treatment with Ang II. Furthermore, candesartan pretreatment significantly promoted the expressions of MYH11, SM22, and smoothelin in Ang II-treated HA-VSMCs (Physique 2B). Whereas, PD123319 did not affect the decline of contractile markers expression in HA-VSMCs treated with Ang II. These data suggest that Ang II regulates HA-VSMCs migration and dedifferentiation via AT1 receptor. Open in a separate window Physique 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 M) for 6 h or PD123319 (5 M) 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 usually 50 m. ** < 0.01 vs. the control group; # < 0.05 vs. the Ang II-treated group (= 3). (B) Western blot analysis of MYH11, SM22, and smoothelin. Histograms show the ratios of MYH11 or SM22 or smoothelin to -tubulin. * < 0.05 vs. the control group; # < 0.05 vs. the Ang II-treated group (= 3). 2.2. RhoA Activation Participates in HA-VSMC Migration and Dedifferentiation Induced by Ang II-AT1R Signaling To assess whether Rho GTPases participate in the regulation of Ang II on HA-VSMC phenotype transition, we analyzed the activation of Rho GTPases by pulldown assay. As shown in Physique 3A, there were no obvious increases in the active style of Rac1 or Cdc42 after Ang II treatment, while the active style of RhoA (RhoA GTP-bound) was significantly increased when cells were treated with Ang II. Candesartan pretreatment exhibited a significant inhibition on RhoA activation, but did not affect the activity of Rac1 or Cdc42 in Ang II-treated HA-VSMCs. In addition, RhoA inhibitor CCG-1423 pretreatment obviously decreased cell migration when compared with the Ang II-treated group (Physique 4A). Different from RhoA inhibition, both Rac1 and Cdc42 inhibition (NSC23766 and ZCL278 pretreatment) exhibited no significant inhibition on cell migration after Ang II treatment (Physique 5A and Physique 6A). Furthermore, we investigated the effects of Rho GTPases inhibition around the expression of VSMC differentiation markers in Ang II-treated HA-VSMCs. As illustrated in Physique 4B, the decline in the expression of MYH11, SM22, and smoothelin was markedly reduced by RhoA inhibition in Ang II-stimulated HA-VSMCs. Nevertheless, in the inhibition of Rac1 and Cdc42, no significant changes on VSMC differentiation markers expression were observed in comparison to the Ang II-treated group (Physique 5B and Physique 6B). These data demonstrate that RhoA activation,.Irbesartan (50 mg/kg/d) and fasudil (30 mg/kg/d) were added to drinking water for consumption by mice. AT1 receptor and then affects VSMC dedifferentiation. < 0.05 and ** < 0.01 vs. the control group (= 3). (C and D) Western blot analysis of MYH11, SM22, and smoothelin. Histograms show the ratios of myosin heavy chain (MYH11) or SM22 or smoothelin to -tubulin. * < 0.05 and ** < 0.01 vs. the control group; # < 0.05 and ## < 0.01 vs. the group treated with 10 nM Ang II; $ < 0.05 vs. the group treated with 100 nM Ang II (= 3). To evaluate the role of AT1 and AT2 receptors on Ang II-mediated cell migration and dedifferentiation, we examined the effects of AT1 or AT2 receptor inhibitor on HA-VSMCs migration and the expressions of MYH11, SM22, and smoothelin after 100 nM Ang II treatment for 24 h. As shown in Figure 2A, pretreatment with the AT1 receptor inhibitor candesartan obviously reduced Ang II-mediated cell migration in HA-VSMCs. In contrast, pretreatment with the AT2 receptor inhibitor PD123319 did not significantly affect HA-VSMCs migration at 24 h treatment with Ang II. Furthermore, candesartan pretreatment significantly promoted the expressions of MYH11, SM22, and smoothelin in Ang II-treated HA-VSMCs (Figure 2B). Whereas, PD123319 did not affect the decline of contractile markers expression in HA-VSMCs treated with Ang II. These data suggest that Ang II regulates HA-VSMCs migration and dedifferentiation via AT1 receptor. Open in a separate window 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 M) for 6 h or PD123319 (5 M) 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 m. ** < 0.01 vs. the control group; # < 0.05 vs. the Ang II-treated group (= 3). (B) Western blot analysis of MYH11, SM22, and smoothelin. Histograms show the ratios of MYH11 or SM22 or smoothelin to -tubulin. * < 0.05 vs. the control group; # < 0.05 vs. the Ang II-treated group (= 3). 2.2. RhoA Activation Participates in HA-VSMC Migration and Dedifferentiation Induced by Ang II-AT1R Signaling To assess whether Rho GTPases participate in the regulation of Ang II on HA-VSMC phenotype transition, we analyzed the activation of Rho GTPases by pulldown assay. As shown in Figure 3A, there were no obvious increases in the active style of Rac1 or Cdc42 after Ang II treatment, while the active style of RhoA (RhoA GTP-bound) was significantly increased when cells were treated with Ang II. Candesartan pretreatment exhibited a significant inhibition on RhoA activation, but did not affect the activity of Rac1 or Cdc42 in Ang II-treated HA-VSMCs. In addition, RhoA inhibitor CCG-1423 pretreatment obviously decreased cell migration when compared with the Ang II-treated group (Figure 4A). Different from RhoA inhibition, both Rac1 and Cdc42 inhibition (NSC23766 and ZCL278 pretreatment) exhibited no significant inhibition on cell migration after Ang II treatment (Figure 5A and Figure 6A). Furthermore, we investigated the effects of Rho GTPases inhibition on the expression of VSMC differentiation markers in Ang II-treated HA-VSMCs. As illustrated in Figure 4B, the decline in the expression of MYH11, SM22, and smoothelin was markedly reduced by RhoA inhibition in Ang II-stimulated HA-VSMCs. Nevertheless, in the inhibition of Rac1 and Cdc42, no significant changes on VSMC.G-actin/F-actin In Vivo Assay Kit (# BK037) and Rac1/Rho/Cdc42 GTPase activity assay kit (# BK030) were purchased from Cytoskeleton, Inc (Denver, CO, USA). 0.05 and ** < 0.01 vs. the control group (= 3). (C and D) Western blot analysis of MYH11, SM22, and smoothelin. Histograms show the ratios of myosin heavy chain (MYH11) or SM22 or smoothelin to -tubulin. * < 0.05 and ** < 0.01 vs. the control group; # < 0.05 and ## < 0.01 vs. the group treated with 10 nM Ang II; $ < 0.05 vs. the group treated with 100 nM Ang II (= 3). To evaluate the role of AT1 and AT2 receptors on Ang II-mediated cell migration and dedifferentiation, we examined the effects of AT1 or AT2 receptor inhibitor on HA-VSMCs migration and the expressions of MYH11, SM22, and smoothelin after 100 nM Ang II treatment for 24 h. As shown in Figure 2A, pretreatment with the AT1 receptor inhibitor candesartan obviously reduced Ang II-mediated cell migration in HA-VSMCs. In contrast, pretreatment with the AT2 receptor inhibitor PD123319 did not significantly affect HA-VSMCs migration at 24 h treatment with Ang II. Furthermore, candesartan pretreatment significantly promoted the expressions of MYH11, SM22, and smoothelin in Ang II-treated HA-VSMCs (Figure 2B). Whereas, PD123319 did not affect the decline of contractile markers expression in HA-VSMCs treated with Ang II. These data suggest that Ang II regulates HA-VSMCs migration and dedifferentiation via AT1 receptor. Open in a separate window 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 M) for 6 h or PD123319 (5 M) 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 m. ** < 0.01 vs. the control group; ESI-09 # < 0.05 vs. the Ang II-treated group (= 3). (B) Western blot analysis of MYH11, SM22, and smoothelin. Histograms show the ratios of MYH11 or SM22 or smoothelin to -tubulin. * < 0.05 vs. the control group; # < 0.05 vs. the Ang II-treated group (= 3). 2.2. RhoA Activation Participates in HA-VSMC Migration and Dedifferentiation Induced by Ang II-AT1R Signaling To assess whether Rho GTPases participate in the regulation of Ang II on HA-VSMC phenotype transition, we analyzed the activation of Rho GTPases by pulldown assay. As shown in Figure 3A, there were no obvious increases in the active style of Rac1 or Cdc42 after Ang II treatment, while the active style of RhoA (RhoA GTP-bound) was significantly increased when cells were treated with Ang II. Candesartan pretreatment exhibited a significant inhibition on RhoA activation, but did not affect the activity of Rac1 or Cdc42 in Ang II-treated HA-VSMCs. In addition, RhoA inhibitor CCG-1423 pretreatment obviously decreased cell migration when compared with the Ang II-treated group (Figure 4A). Different from RhoA inhibition, both Rac1 and Cdc42 inhibition (NSC23766 and ZCL278 pretreatment) exhibited no significant inhibition on cell.However, the effects of different Rho GTPase members on Ang II-mediated smooth muscle migration and dedifferentiation in human cell lines and the mechanisms behind these effects are not well understood, though we recently revealed a novel mechanism by which Ang II regulates VSMC proliferation by Rho-specific guanine nucleotide dissociation inhibitor (RhoGDI) protein stability [12]. 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. < 0.05 and ** < 0.01 vs. the control group (= 3). (C and D) Western blot analysis of MYH11, SM22, and smoothelin. Histograms show the ratios of myosin heavy chain (MYH11) or SM22 or smoothelin to -tubulin. * < 0.05 and ** < 0.01 vs. the control group; # < 0.05 and ## < 0.01 vs. the group treated with 10 nM Ang II; $ < 0.05 vs. the group treated with 100 nM Ang II (= 3). To evaluate the part of AT1 and AT2 receptors on Ang II-mediated cell migration and dedifferentiation, we examined the effects of AT1 or AT2 receptor inhibitor on HA-VSMCs migration and the expressions of MYH11, SM22, and smoothelin after 100 nM Ang II treatment for 24 h. As demonstrated in Number 2A, pretreatment with the AT1 receptor inhibitor candesartan obviously reduced Ang II-mediated cell migration in HA-VSMCs. In contrast, pretreatment with the AT2 receptor inhibitor PD123319 did not significantly affect HA-VSMCs migration at 24 h treatment with Ang II. Furthermore, candesartan pretreatment significantly advertised the expressions of MYH11, SM22, and smoothelin in Ang II-treated HA-VSMCs (Number ESI-09 2B). Whereas, PD123319 did not affect the decrease of contractile markers manifestation in HA-VSMCs treated with Ang II. These data suggest that Ang II regulates HA-VSMCs migration and dedifferentiation via AT1 receptor. Open in a separate window Number 2 The effects of Ang II receptor inhibitors on cell migration and the manifestation of contractile marker proteins in HA-VSMCs. Cells were pretreated with candesartan (5 M) for 6 h or PD123319 (5 M) 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 recognized by wound-healing and Transwell assays. Histograms display the quantification of the wound healing and Transwell assay results. The scar pub is definitely 50 m. ** < 0.01 vs. the control group; # < 0.05 vs. the Ang II-treated group (= 3). (B) Western blot analysis of MYH11, SM22, and smoothelin. Histograms display the ratios of MYH11 or SM22 or smoothelin to -tubulin. * < 0.05 vs. the control group; # < 0.05 vs. the Ang II-treated group (= 3). 2.2. RhoA Activation Participates in HA-VSMC Migration and Dedifferentiation Induced by Ang II-AT1R Signaling To assess whether Rho GTPases participate in the rules of Ang II on HA-VSMC phenotype transition, we analyzed the activation of Rho GTPases by pulldown assay. As demonstrated in Number 3A, there were no obvious raises in the active style of Rac1 or Cdc42 after Ang II treatment, while the active style of NNT1 RhoA (RhoA GTP-bound) was significantly improved when cells were treated with Ang II. Candesartan pretreatment exhibited a significant inhibition on RhoA activation, but did not affect the activity of Rac1 or Cdc42 in Ang II-treated HA-VSMCs. In addition, RhoA inhibitor CCG-1423 pretreatment obviously decreased cell migration when compared with the Ang II-treated group (Number 4A). Different from RhoA inhibition, both Rac1 and Cdc42 inhibition (NSC23766 and ZCL278 pretreatment) exhibited no significant inhibition on cell migration after Ang II treatment (Number 5A and Number 6A). Furthermore, we investigated the effects of Rho GTPases inhibition within the manifestation of VSMC differentiation markers in Ang II-treated HA-VSMCs. As illustrated in Number 4B, the decrease in the manifestation of MYH11, SM22, and smoothelin was markedly reduced by RhoA inhibition in Ang II-stimulated HA-VSMCs. However, in the inhibition of Rac1 and Cdc42, no significant changes on VSMC differentiation markers manifestation were observed in comparison to the Ang II-treated group (Number 5B and Number 6B). These data demonstrate that RhoA activation, but not Rac1 and Cdc42 activity, stimulated by Ang II-AT1 receptor signaling participates in.* < 0.05, vs. shown to be dependent on AT1 receptor and RhoA/ROCK pathway. Summary: This study discloses a novel mechanism by which Ang II regulates RhoA/ROCK signaling and actin polymerization via AT1 receptor and then affects VSMC dedifferentiation. < 0.05 and ** < ESI-09 0.01 vs. the control group (= 3). (C and D) Western blot analysis of MYH11, SM22, and smoothelin. Histograms display the ratios of myosin weighty chain (MYH11) or SM22 or smoothelin to -tubulin. * < 0.05 and ** < 0.01 vs. the control group; # < 0.05 and ## < 0.01 vs. the group treated with 10 nM Ang II; $ < 0.05 vs. the group treated with 100 nM Ang II (= 3). To evaluate the part of AT1 and AT2 receptors on Ang II-mediated cell migration and dedifferentiation, we examined the effects of AT1 or AT2 receptor inhibitor on HA-VSMCs migration and the expressions of MYH11, SM22, and smoothelin after 100 nM Ang II treatment for 24 h. As demonstrated in Number 2A, pretreatment with the AT1 receptor inhibitor candesartan obviously reduced Ang II-mediated cell migration in HA-VSMCs. In contrast, pretreatment with the AT2 receptor inhibitor PD123319 did not significantly affect HA-VSMCs migration at 24 h treatment with Ang II. Furthermore, candesartan pretreatment significantly advertised the expressions of MYH11, SM22, and smoothelin in Ang II-treated HA-VSMCs (Number 2B). Whereas, PD123319 did not affect the decrease of contractile markers manifestation in HA-VSMCs treated with Ang II. These data suggest that Ang II regulates HA-VSMCs migration and dedifferentiation via AT1 receptor. Open in a separate window Number 2 The effects of Ang ESI-09 II receptor inhibitors on cell migration and the manifestation of contractile marker proteins in HA-VSMCs. Cells were pretreated with candesartan (5 M) for 6 h or PD123319 (5 M) 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 recognized by wound-healing and Transwell assays. Histograms display the quantification of the wound healing and Transwell assay results. The scar pub is definitely 50 m. ** < 0.01 vs. the control group; # < 0.05 vs. the Ang II-treated group (= 3). (B) Western blot analysis of MYH11, SM22, and smoothelin. Histograms display the ratios of MYH11 or SM22 or smoothelin to -tubulin. * < 0.05 vs. the control group; # < 0.05 vs. the Ang II-treated group (= 3). 2.2. RhoA Activation Participates in HA-VSMC Migration and Dedifferentiation Induced by Ang II-AT1R Signaling To assess whether Rho GTPases participate in the rules of Ang II on HA-VSMC phenotype transition, we analyzed the activation of Rho GTPases by pulldown assay. As demonstrated in Number 3A, there were no obvious raises in the active style of Rac1 or Cdc42 after Ang II treatment, while the active style of RhoA (RhoA GTP-bound) was significantly improved when cells were treated with Ang II. Candesartan pretreatment exhibited a significant inhibition on RhoA activation, but did not affect the activity of Rac1 or Cdc42 in Ang II-treated HA-VSMCs. In addition, RhoA inhibitor CCG-1423 pretreatment obviously decreased cell migration when compared with the Ang II-treated group (Number 4A). Different from RhoA inhibition, both Rac1 and Cdc42 inhibition (NSC23766 and ZCL278 pretreatment) exhibited no significant inhibition on cell migration after Ang II treatment (Number 5A and Number 6A). Furthermore, we investigated the effects of Rho GTPases inhibition within the manifestation of VSMC differentiation markers in Ang II-treated HA-VSMCs. As illustrated in Number 4B, the decrease in the manifestation of MYH11, SM22, and smoothelin was markedly reduced by RhoA inhibition in Ang II-stimulated HA-VSMCs. However, in the ESI-09 inhibition of Rac1 and Cdc42, no significant changes on VSMC differentiation markers manifestation were observed in comparison to the Ang II-treated group (Number 5B and Number 6B). These data demonstrate.