Korean J Physiol Pharmacol.  2009 Oct;13(5):385-392. 10.4196/kjpp.2009.13.5.385.

Downregulation of Angiotensin II-Induced 12-Lipoxygenase Expression and Cell Proliferation in Vascular Smooth Muscle Cells from Spontaneously Hypertensive Rats by CCL5

• Kim JH
• Kim HS

• KMID: 1982573
• DOI: http://doi.org/10.4196/kjpp.2009.13.5.385

Abstract

Angiotensin II (Ang II) plays an important role in vascular hypertension. The role of the chemokine CCL5 on Ang II-induced activities in vascular smooth muscle cells (VSMCs) has not been studied. In this study, we elucidated the effect of CCL5 on Ang II-induced 12-lipoxygenase (LO) expression and cell proliferation in spontaneously hypertensive rats (SHR) VSMCs. CCL5 decreased Ang II-induced 12-LO mRNA expression and protein production, and it increased Ang II type 2 (AT2) receptor expression in SHR VSMCs. The inhibitory effect of CCL5 on Ang II-induced 12-LO mRNA expression was mediated through the AT2 receptor. Although treatment of CCL5 alone induced SHR VSMCs proliferation, CCL5 inhibited Ang II-induced VSMCs proliferation and PD123,319, an AT2 receptor antagonist, blocked the inhibitory effect of CCL5 on Ang II-induced VSMCs proliferation. Phosphorylation of p38 was detected in VSMCs treated with Ang II or CCL5 alone. But, decrease of p38 phosphorylation was detected in VSMCs treated with Ang II and CCL5 simultaneously (Ang II/CCL5) and PD123,319 increased p38 phosphorylation in VSMCs treated with Ang II/CCL5. Therefore, these results suggest that the inhibitory effect of CCL5 on Ang II-induced VSMCs proliferation is mediated by the AT2 receptor via p38 inactivation, and CCL5 may play a beneficial role in Ang II-induced vascular hypertension.

Keyword

Angiotensin II; Angiotensin II type 2 receptor; Cell proliferation; Chemokine CCL5; 12-lipoxygenase

MeSH Terms

Angiotensin II
Angiotensins
Arachidonate 12-Lipoxygenase
Cell Proliferation
Chemokine CCL5
Down-Regulation
Hypertension
Muscle, Smooth, Vascular
Phosphorylation
Rats, Inbred SHR
Receptor, Angiotensin, Type 2
RNA, Messenger
Angiotensin II
Angiotensins
Arachidonate 12-Lipoxygenase
Chemokine CCL5
RNA, Messenger
Receptor, Angiotensin, Type 2

Figure

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  Fig. 1. Costitutive expression of CCL5 mRNA in SHR VSMCs is lower than the expression in WKY VSMCs, and Ang II inhibits CCL5 mRNA expression and protein production in SHR VSMCs. (A) After total RNAs were isolated from SHR or WKY VSMCs, real-time PCR was performed. Bars represent means±SD from three independent experiments. ∗p<0.05 vs. WKY VSMCs. (B, C) SHR VSMCs were untreated or treated with Ang II (0.1 μmol/l). After total RNAs and cell supernatants were isolated, real time PCR (B) and ELISA (C) were performed. Bars represent means±SD from three independent experiments. ∗p<0.05 vs. untreated VSMCs.

• 

  Fig. 2. CCL5 inhibits Ang II-induced 12-LO mRNA expression in SHR VSMCs. (A) SHR VSMCs were untreated or treated with Ang II (0.1 μmol/l) and/or CCL5 (100 ng/ml) for 2 h. After total RNAs were isolated, real-time PCR was performed. Bars represent means±SD from three independent experiments. ∗p<0.05 vs. VSMCs treated with Ang II. (B) SHR VSMCs were untreated or treated with Ang II (0.1 μmol/l) and/or CCL5 (100 ng/ml) for 2 h. Cell lysates were prepared and separated on 10% SDS-polyacrylamide gels and then immunoblotted with 12-LO antibody. The data shown are representative of three independent experiments. (C) Dose response of Ang II-induced 12-LO mRNA expression in SHR VSMCs to CCL5. SHR VSMCs were untreated (NT) or treated with 0, 10, 50, 100, or 200 ng/ml of CCL5 for 2 h. After total RNAs were isolated, real-time PCR was performed. Bars represent means±SD from three independent experiments.

• 

  Fig. 3. The inhibitory effect of CCL5 on Ang II-induced 12-LO mRNA expression is mediated through the AT2 receptor in SHR VSMCs. (A, B) SHR VSMCs were untreated or treated with Ang II (0.1 μmol/l) and/or CCL5 in the presence or absence of losartan (AT1 receptor antagonist, 10 μmol/l, A) or PD123,319 (AT2 receptor antagonist, 10 μmol/l, B) for 2 h and total RNAs were analyzed by real-time PCR. Bars represent means±SD from three independent experiments. ap<0.05 vs. VSMCs treated with Ang II. bp<0.05 vs. VSMCs treated with CCL5, cp<0.05 vs. VSMCs treated with Ang II and CCL5 simultaneously (Ang II/CCL5). (C, D) SHR VSMCs were plated on 24-well plates, grown to 90% confluence and then transfected with AT2 receptor siRNA oligomers (50 nmol/l). VSMCs were then untreated or treated with AngII (0.1 μmol/l) for 2 h for RT-PCR (C) and VSMCs were untreated or treated with AngII (0.1 μmol/l) and/or CCL5 (100 ng/ml) for 2 h, and total RNAs were analyzed by real-time PCR (D). Non TF: non-transfected VSMCs, TF: transfected VSMCs. Bars represent means±SEM from two independent experiments. The data are representative of two independent experiments.

• 

  Fig. 4. CCL5 increases the mRNA expression and production of AT2 receptor in SHR VSMCs. SHR VSMCs were untreated (NT) or treated with CCL5 (100 ng/ml) and or Ang II (0.1 μmol/l) for 2 h. Then, total RNAs for real time PCR (A) and cell lysates for protein detection (B) were isolated. Cell lysates were separated on 10% SDS-polyacrylamide gels and then immunoblotted with AT2 receptor antibody. Bars represent means±SD from four independent experiments ∗p<0.05 vs. untreated SHR VSMCs. The data shown are representative of three independent experiments.

• 

  Fig. 5. CCL5 induces SHR VSMCs proliferation, but simultaneous treatment of Ang II and CCL5 inhibits SHR VSMCs proliferation. (A) SHR VSMCs were treated with AngII (0.1 μmol/l) and/or CCL5 (100 ng/ml) for 24 h in medium containing [3H]-thymidine (1 μCi/ml). [3H]-thymidine incorporation is shown on the Y-axis. Bars represent means±SD from three independent experiments. ∗p<0.05 vs. VSMCs treated with Ang II. (B) SHR VSMCs were untreated (NT) or treated with Ang II (0.1 μmol/l) and/or CCL5 (100 ng/ml) for 2 h. Total RNAs were isolated and real-time PCR for ODC mRNA expression was performed. Bars represent means± SD from three independent experiments. ∗p<0.05 vs. VSMCs treated with Ang II.

• 

  Fig. 6. The inhibitory effect of CCL5 on Ang II-induced VSMCs proliferation is mediated by the AT2 receptor. (A, B) SHR VSMCs were treated with Ang II (0.1 μmol/l) and/or CCL5 (100 ng/ml) in the presence or absence of losartan (AT1 receptor antagonist, 10 μmol/l, A) or PD123,319 (AT2 receptor antagonist, 10 μmol/l, B) for 24 h in medium containing [3H]-thymidine (1 μCi/ml). [3H]-thymidine incorporation is shown on the Y-axis. Bars represent means±SD from three independent experiments. ap<0.05 vs. VSMCs treated with Ang II. bp<0.05 vs. VSMCs treated with CCL5. ∗p<0.05 vs. VSMCs treated with Ang II/CCL5. (C) SHR VSMCs were plated on 24-well plates, grown to 90% confluence and then transfected with AT2 receptor siRNA oligomers (50 nmol/l). VSMCs were then untreated or treated Ang II (0.1 μmol/l) and/or CCL5 (100 ng/ml) for 24 h in medium containing [3H]-thymidine (1 μCi/ml). [3H]-thymidine incorporation is shown on the Y-axis. Bars represent means±SD from three independent experiments.

• 

  Fig. 7. The inhibitory effect of Ang II/CCL5 on VSMCs proliferation is mediated by the AT2 receptor via p38 inactivation. (A) SHR VSMCs were untreated (NT) or pretreated with PD169316 for 30 min. Then, cells were treated with Ang II (0.1 μmol/l) and/or CCL5 (100 ng/ml) in the presence or absence of PD123,319 (10 μmol/l) for 24 h in medium containing [3H]-thymidine (1 μCi/ml). [3H]-thymidine incorporation is shown on the Y-axis. Bars represent means±SD from three independent experiments. ap<0.05 vs. VSMCs treated with Ang II. bp<0.05 vs. VSMCs treated with Ang II/CCL5. cp<0.05 vs. VSMCs treated with PD123,319 and Ang II/CCL5. (B) SHR VSMCs were untreated or treated with Ang II (0.1 μmol/l) and/or CCL5 (100 ng/ml) in the presence or absence of PD123,319 (10 μmol/l) for 2 h. Cell lysates were separated on 10% SDS-polyacrylamide gels and then immunoblotted with phospho-p38 or p38 antibody. Data shown are representative of three independent experiments.

Reference

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