Myofibrillogenesis Regulator-1 in Smooth Muscle Cells Modulates Inflammation Signaling Pathways via Regulating ROCK1 Ubiquitination and Degradation to Impact Aortic Dissection

Figures

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  Figure 1. Differences in structural and inflammatory expression levels in the aorta between AD and non-AD groups. (A) HE staining and MASSON staining of human aorta in AD and Ctrl groups. (B) Immunofluorescence staining and (C) quantification of IL-1β (red), IL-6 (red) and TNF-α (red) in human aortas in AD or Ctrl groups (unpaired two-tailed Student’s t-test with Welch’s correction for IL-1β, IL-6 and TNF-α). Scale bar = 75 μm. Each datapoint represents an individual human. n=10 human aortas (AD or Ctrl). (D) Western blot analysis and (E) quantification of pNF-kappaB p65, IL-1β, IL-6, and TNF-α in human aortas with AD or Ctrl (unpaired two-tailed Student’s t-test with Welch’s correction for pNF-kappaB p65, IL-1β, IL-6 and TNF-α). β-actin was used as a loading control. Data are shown as mean ± SEM. Each datapoint represents an individual human. n=10 human aortas (AD or Ctrl). All data are based on biological replicates. The duplication occurred in Figures 1D and 2C E (β-actin) because the experimental conditions and sample sources for these groups were consistent. *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 2. The expression levels of MR-1 and ROCK1 were found to be markedly upregulated in the aortas of the AD group. (A) Immunohistochemistry and (B) quantification of MR-1 (brown), ROCK1 (brown) in human aortas from AD and Ctrl groups (unpaired two-tailed Student’s t-test with Welch’s correction for MR-1, ROCK1). Each datapoint represents an individual human. n=10 human aortas (AD or Ctrl). (C) Western blot analysis and (D) quantification of ROCK, MR-1, α-SMA, OPN, MMP2 and MMP9 in human aortas with AD or Ctrl (unpaired two-tailed Student’s t-test with Welch’s correction for MR-1,ROCK). β-actin was used as a loading control. Data are shown as mean ± SEM. Each datapoint represents an individual human. n=10 human aortas (AD or Ctrl). All data are based on biological replicates. (E) Immunofluorescence was used to identify the purity of smooth muscle cells. Scale bar = 250 μ m. (F) Western blot analysis and (G) quantification of MR-1, ROCK1 protein expression levels and inflammatory pathway activation levels in AD or Ctrl vSMCs (one-way ANOVA followed by Bonferroni post hoc test). n=3. Data are shown as mean±SEM. All data are based on biological replicates. The duplication occurred in…

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  Figure 3. Continued.

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  Figure 3. Inhibition of MR-1 reduces vSMCs inflammatory expression, inhibits vSMCs phenotypic transformation and ECM degradation. (A) Western blotting and quantification (B) of α-SMA, OPN, MMP2, MMP9 and MR-1 in the presence or absence of MR-1 siRNA in human vSMCs cultured in angiotensin II (1μM) for 24 h (one-way ANOVA followed by Bonferroni post hoc test). β-actin was used as a loading control. n=3. (C) AD smooth muscle cell model with or without MR-1 siRNA and Western blotting and quantification (D) of NF-kappaB p65 phosphorylation activation and levels of inflammatory factors IL-1β, IL-6, and TNF-α release in the MR-1 siRNA NC group (one-way ANOVA followed by Bonferroni post hoc test). β-actin was used as a loading control. n=3. (E) Representative images of cellular immunofluorescence detection of NF-kappaB p65 (green) in angiotensin II-treated (1μM) human vSMCs in the presence or absence of MR-1 siRNA. Scale bar = 50 μm. (F) The mRNA levels of inflammatory factors IL-1β, IL-6, and TNF-α release in the presence or absence of MR-1 siRNA in human vSMCs (one-way ANOVA followed by Bonferroni post hoc test). n=3. Data are shown as mean±SEM. All data are based on biological replicates. The…

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  Figure 4. Continued.

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  Figure 4. Inhibition of ROCK1 reduces vSMCs inflammatory expression, inhibits vSMCs phenotypic transformation and ECM degradation. (A) Western blotting and quantification (B) of α-SMA, OPN, MMP9 and ROCK1 in the presence or absence of ROCK1 siRNA in human vSMCs cultured in angiotensin II (1μM) for 24 h (one-way ANOVA followed by Bonferroni post hoc test). β-actin was used as a loading control. n=3. (C) AD smooth muscle cell model with or without ROCK1 siRNA and Western blotting and quantification (D) of NF-kappaB p65 phosphorylation activation and levels of inflammatory factors IL-1β, IL-6, and TNF-α release in the ROCK1 siRNA NC group (one-way ANOVA followed by Bonferroni post hoc test). β-actin was used as a loading control. n=3. (E) Representative images of cellular immunofluorescence detection of NF-kappaB p65 (green) in angiotensin II-treated (1μM) human vSMCs in the presence or absence of ROCK1 siRNA. Scale bar = 50 μm. (F) The mRNA levels of inflammatory factors IL-1β, IL-6, and TNF-α release in the presence or absence of ROCK1 siRNA in human vSMCs (one-way ANOVA followed by Bonferroni post hoc test). n=3. Data are shown as mean±SEM. All data are based on biological replicates. The…

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  Figure 5. MR-1 Affects AD Progression by Regulating the ROCK1-NF-kappaB p65 Signaling Pathway. (A and B) Co-localization of MR-1 (green) with ROCK1 (red) in human vSMCs induced by angiotensinII for 24 hours. Scale bar= 50 μm. (C and D) Protein immunoblotting and quantitative analysis of ROCK1 upon inhibition of MR-1 and (E and F) protein immunoblotting and quantitative analysis of MR-1 upon inhibition of ROCK1 in human vSMCs induced by angiotensinII for 24 hours (one-way ANOVA followed by Bonferroni post hoc test). β-actin was used as a loading control. n=3. (G) RT-PCR was performed to detect mRNA levels of ROCK1 in human vSMCs treated with angiotensinII in the presence or absence of MR-1 siRNA (one-way ANOVA followed by Bonferroni post hoc test). n=3. Data are shown as mean ± SEM. All data are based on biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 6. Increased expression of MR-1 inhibits ubiquitination and degradation of ROCK1. (A and B) Western blotting was performed to detect protein levels of ROCK1 in human vSMCs treated with cycloheximide (CHX, 10 μM) and angiotensin II for the indicated times in the presence or absence of MR-1 siRNA (one-way ANOVA followed by Bonferroni post hoc test). β-actin was used as a loading control. n=3. (C and D) After preincubation with MG132 (10 μM) or chloroquine (CQ, 10 μM) for 30 minutes, Western blotting was performed to detect the protein level of ROCK1 in angiotensin II -treated human vSMCs in the presence or absence of MR-1 siRNA (one-way ANOVA followed by Bonferroni post hoc test). β-actin was used as a loading control. n=3. (E) Co-IP was performed to detect the ubiquitination level of ROCK1 in angiotensin II -treated human vSMCs in the presence or absence of MR-1 siRNA. β-actin was used as a loading control. n=3. Data are shown as mean ± SEM. All data are based on biological replicates. *P < 0.05.