Angiotensin III Induces JAK2/STAT3 Leading to IL-6 Production in Rat Vascular Smooth Muscle Cells

Figures

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  Figure 1. Effect of concentration and time on angiotensin (Ang) II- and Ang III-mediated STAT3 protein phosphorylation in Wistar vascular smooth muscle cells (VSMCs). Quiescent Wistar VSMCs were treated for (A) 10 min with either Ang II or Ang III at concentrations ranging from 0.1 nM to 1000 nM or (B) with 100 nM Ang II or 100 nM Ang III for 1 min to 30 min. Phosphorylated-STAT3 protein levels were measured by Western blot analysis using a specific antibody for the phosphorylated form of STAT3. Protein loading was visualized using the non-phosphorylated STAT3 antibody. The data were analyzed by densitometry and the amount of phosphorylation was calculated as the fold-increase above basal in the presence of vehicle. Each value represents the mean ± SEM of preparations of VSMCs isolated from at least five Wistar rats. * Denotes p < 0.05 as compared to basal levels of STAT3 expression in VSMCs for Ang III. # Denotes p < 0.05 as compared to STAT3 basal levels in VSMCs for Ang II.

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  Figure 2. Effect of concentration and time on Ang III-mediated STAT3 protein phosphorylation in SHR VSMCs. Quiescent SHR VSMCs were treated for (A) 10 min with Ang III at concentrations ranging from 0.1 nM to 1000 nM or (B) with 100 nM Ang II or 100 nM Ang III for 1 min to 30 min. The data shown in Figure 2A,B are the same as the data in Figure 1A,B, respectively, for the Wistar Ang III data. Phosphorylated-STAT3 protein levels were measured by Western blot analysis using a specific antibody for the phosphorylated form of STAT3. Protein loading was visualized using the non-phosphorylated STAT3 antibody. The data were analyzed by densitometry and the amount of phosphorylation was calculated as the fold-increase above basal in the presence of vehicle. Each value represents the mean ± SEM of preparations of VSMCs isolated from at least five Wistar or SHRs. * Denotes p < 0.05 as compared to basal levels of STAT3 expression in VSMCs isolated from Wistar rats. # Denotes p < 0.05 as compared to STAT3 basal levels in VSMCs isolated from SHRs.

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  Figure 3. Effects of AG490, Losartan, PD123319 on Ang III-induced STAT3 Phosphorylation in Wistar VSMCs. Quiescent Wistar VSMCs were pretreated with either 50 µM AG490 (A) or 10 µM Losartan (Los) or 10 µM PD123319 (B) for 15 min. Subsequently, the cells were treated with or without 100 nM Ang III for 10 min. Phosphorylated-STAT3 protein levels were measured by Western blot analysis using a specific antibody for the phosphorylated form of STAT3. Protein loading was visualized using the non-phosphorylated STAT3 antibody. The data were analyzed by densitometry and the amount of phosphorylation was calculated as the fold-increase above basal in the presence of vehicle. The vehicle for AG490 was DMSO; incubation with Ang III was also done in the presence of DMSO. Each value represents the mean ± SEM of preparations of VSMCs isolated from at five or more Wistar rats. * Denotes p < 0.05 as compared to basal levels of STAT3 expression in VSMCs isolated from Wistar rats.

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  Figure 4. Effect of time on Ang II- and Ang III-induced IL-6 secretion in Wistar and SHR VSMCs. Quiescent VSMCs isolated from (A) Wistar rats treated with Ang II or Ang III, and (B) SHRs treated with Ang II or Ang III were treated with 100 nM Ang II or 100 nM Ang III for 3 h to 48 h. Panel (C) compares Ang III-mediated IL-6 secretion from Wistar and SHR VSMCS. Panel (D) compares Ang II-mediated IL-6 secretion from Wistar and SHR VSMCs. Secreted levels of IL-6, induced by Ang II and Ang III, were calculated as the fold above basal and were analyzed by an ELISA method. Each value represents the mean ± SEM of preparations of VSMCs isolated from four or more SHRs or Wistar rats. * Denotes p < 0.05 as compared to basal levels of IL-6 secretion in VSMCs treated with Ang III. # Denotes p < 0.05 as compared to basal levels of IL-6 secretion in VSMCs treated with Ang II. ^ Denotes p < 0.05 as compared between the effects of Ang II or Ang III in stimulation of IL-6 secretion in VSMCs isolated from SHRs versus Wistar rats.

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  Figure 5. Effects of AG490, Losartan, PD123319 on Ang III-induced IL-6 secretion in Wistar VSMCs. Quiescent Wistar VSMCs were pretreated with either 50 µM AG490 (A) or 10 µM Losartan (Los) or 10 µM PD123319 (B) for 15 min. Subsequently, the cells were treated with or without 100 nM Ang III for 3 h. Secreted levels of IL-6, induced Ang III from Wistar VSMCs, were calculated as the fold above basal and were analyzed by an ELISA method. The vehicle for AG490 was DMSO; incubation with Ang III was also done in the presence of DMSO. Each value represents the mean ± SEM of preparations of VSMCs isolated from four or more Wistar rats. * Denotes p < 0.05 as compared to basal levels of IL-6 secretion from VSMCs isolated from Wistar rats.

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  Figure 6. Effect of time on Ang II- and Ang III-induced IL-6 mRNA expression in Wistar and SHR VSMCs. Quiescent VSMCs isolated from (A) Wistar rats treated with Ang II or Ang III, and (B) SHRs treated with Ang II or Ang III were incubated with 100 nM Ang II or 100 nM Ang III for 1 h to 48 h. Panel (C) compares Ang III-mediated IL-6 mRNA expression from Wistar and SHR VSMCS. Panel (D) compares Ang II-mediated IL-6 mRNA expression from Wistar and SHR VSMCs. IL-6 mRNA expression was analyzed by quantitative PCR. The amount of Ang II- and Ang III-stimulated IL-6 mRNA expression was calculated as the fold-increase above basal. Each value represents the mean ± SEM of preparations of VSMCs isolated from four or more Wistar rats or SHRs. * Denotes p < 0.05 as compared to basal levels of IL-6 mRNA expression in Wistar VSMCs treated with Ang III or Ang II. # Denotes p < 0.05 as compared to basal levels of IL-6 mRNA expression in SHR VSMCs treated with Ang II or Ang III. ^ Denotes p < 0.05 as compared between the effects of Ang II or Ang III in stimulation of IL-6 mRNA expression in VSMCs isolated from Wistar rats versus SHRs.

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  Figure 7. Effects of AG490, Losartan, PD123319 on Ang III-induced IL-6 mRNA expression in Wistar VSMCs. Quiescent Wistar VSMCs were pretreated with either 50 µM AG490 (A) or 10 µM Losartan (Los) or 10 µM PD123319 (B) for 15 min. Subsequently, the cells were treated with or without 100 nM Ang III for 3 h. IL-6 mRNA expression was analyzed by quantitative PCR. The vehicle for AG490 was DMSO; incubation with Ang III was also done in the presence of DMSO. Each value represents the mean ± SEM of preparations of VSMCs isolated from four or more Wistar rats. * Denotes p < 0.05 as compared to basal levels of IL-6 mRNA expression from VSMCs isolated from Wistar rats.