Korean Circ J.  2019 Jul;49(7):615-626. 10.4070/kcj.2018.0379.

Angiotensin II Type 1 Receptor Blocker, Fimasartan, Reduces Vascular Smooth Muscle Cell Senescence by Inhibiting the CYR61 Signaling Pathway

• Kim I
• Park CS
• Lee HY

• KMID: 2450393
• DOI: http://doi.org/10.4070/kcj.2018.0379

Abstract

BACKGROUND AND OBJECTIVES
Angiotensin II (Ang II) has been suggested to accelerate vascular senescence, however the molecular mechanism(s) remain unknown.
METHODS
We cultured human coronary artery smooth muscle cells (hCSMCs) and treated Ang II and/or fimasartan. Or we transfected adenoviral vectors expressing CYR61 (Ad-CYR61) or antisense CYR61 (Ad-As-CYR61). Cellular senescence was evaluated senescence-associated β-galactosidase (SA-β-gal) assay. The molecular mechanisms were investigated real-time PCR and western blots.
RESULTS
SA-β-gal-positive cells significantly increased in Ang II-treated hCSMCs (5.77±1.43-fold compared with the control). The effect of Ang II was significantly attenuated by pretreatment with the Ang II type 1 receptor blocker, fimasartan (2.00±0.92-fold). The expression of both p53 and p16 senescence regulators was significantly increased by Ang II (p53: 1.39±0.17, p16: 1.19±0.10-fold vs. the control), and inhibited by fimasartan. Cysteine-rich angiogenic protein 61 (CYR61) was rapidly induced by Ang II. Compared with the control, Ad-CYR61-transfected hCSMCs showed significantly increased SA-β-gal-positive cells (3.47±0.65-fold). Upon transfecting Ad-AS-CYR61, Ang II-induced senescence (3.74±0.23-fold) was significantly decreased (1.77±0.60-fold). p53 expression by Ang II was significantly attenuated by Ad-AS-CYR61, whereas p16 expression was not regulated. Ang II activated ERK1/2 and p38 MAPK, which was significantly blocked by fimasartan. ERK and p38 inhibition both regulated Ang II-induced CYR61 expression. However, p53 expression was only regulated by ERK1/2, whereas p16 expression was only attenuated by p38 MAPK.
CONCLUSIONS
Ang II induced vascular senescence by the ERK/p38 MAPK-CYR61 pathway and ARB, fimasartan, protected against Ang II-induced vascular senescence.

Keyword

Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Coronary vessels; Cellular Senescence

MeSH Terms

Aging
Angiotensin II Type 1 Receptor Blockers
Angiotensin II*
Angiotensins*
Blotting, Western
Cell Aging*
Coronary Vessels
Humans
Muscle, Smooth, Vascular*
Myocytes, Smooth Muscle
p38 Mitogen-Activated Protein Kinases
Real-Time Polymerase Chain Reaction
Receptor, Angiotensin, Type 1*
Angiotensin II
Angiotensin II Type 1 Receptor Blockers
Angiotensins
Receptor, Angiotensin, Type 1
p38 Mitogen-Activated Protein Kinases

Figure

• 

  Figure 1 Ang II induces cellular senescence in hCSMCs, whereas Fima inhibits it. (A) Response of SA-β-gal positive cells following treatment with Ang II (1–100 nM) for 7 days. (B) Cellular senescence induced by Ang II was near completely blocked by pretreatment with ARB, Fima (1 µM). Scatter plots with bar show data from 4 independent experiments. Values are given as mean±standard deviation (n=4). Ang II = angiotensin II; ARB = Ang II type 1 receptor blocker; Fima = fimasartan; hCSMC = human coronary artery smooth muscle cell; SA-β-gal = senescence-associated β-galactosidase. *p<0.01, †p<0.0001.

• 

  Figure 2 Ang II induces p53 and p16 expression, whereas Fima inhibits it. hCSMCs were treated with Ang II at 100 nM for 4 hours, and/or pretreated Fima at 1 μM for 2 hours. (A) Western blot and (B) real-time PCR analyses showed expression of p53 and p16 after Ang II treatment. Scatter plots with bar show data from 3 independent experiments. Values are given as mean±standard deviation (n=3). Ang II = angiotensin II; Fima = fimasartan; hCSMC = human coronary artery smooth muscle cell; PCR = polymerase chain reaction. *p<0.001, †p<0.0001.

• 

  Figure 3 Ang II induces CYR61 mRNA expression in hCSMCs, whereas Fima inhibit it. CYR61 mRNA expression was detected by quantitative real-time PCR. (A) CYR61 mRNA expression was increased by following Ang II treatment in concentration-dependent manners (1–100 nM, 120 minutes). (B) Increased CYR61 mRNA expression induced by Ang II at 100 nM was continued for 240 minutes and peaked at 120 minutes. (C) Increased expression of CYR61 mRNA induced by Ang II at 100 nM for 120 minutes was significantly inhibited by pretreated Fima (1 μM). Bar graphs show data from 3 independent experiments. Values are given as mean±standard deviation (n=3). Ang II = angiotensin II; AT1R = angiotensin II type 1 receptor; CYR61 = cysteine-rich angiogenic protein 61; Fima = fimasartan; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; hCSMC = human coronary artery smooth muscle cell. *p<0.05, †p<0.01.

• 

  Figure 4 CYR61 regulates cellular senescence in hCSMCs. hCSMCs were transfected with adenoviral vectors at 50 MOI for 4 hours for induction or suppression CYR61, and control were transfected with Ad-GFP. (A) Induction of CYR61 by transfection with Ad-CYR61 significantly increased SA-β-Gal (+) hCSMCs. (B) However, suppression of CYR61 by transfection with Ad-As-CYR61 decreased SA-β-Gal (+) cells after Ang II treatment (100 nM). Scatter plots with bar show data from 4 independent experiments. Values are given as mean±standard deviation (n=4). Ad-CYR61 = adenoviral vectors expressing CYR61; Ad-As-CYR61 = adenoviral vectors expressing antisense CYR61; Ad-GFP = adenoviral vector expressing green fluorescence proteins; Ang II = angiotensin II; CYR61 = cysteine-rich angiogenic protein 61; hCSMC = human coronary artery smooth muscle cell; SA-β-Gal = senescence-associated β-galactosidase. *p<0.001, †p<0.0001.

• 

  Figure 5 Ang II-CYR61 dependent cellular senescence was mediated by the p53-dependent pathway, but not by the p16-dependent pathway. hCSMCs were transfected with the indicated adenoviral vectors for 4 hours. Control were transfected with Ad-GFP. (A, B) CYR61 and p53 expressions were increased in transfection with Ad-CYR61 (50 MOI). In contrast, p16 expression was not changed. Scatter plots with bar show data from 4 independent experiments. Values are given as mean±standard deviation (n=4). (C) CYR61-induced p53 expressions were increased in a MOI-dependent manner. (D, E) CYR61 inhibition by Ad-AS-CYR61 (50 MOI) significantly inhibited p53 expression, whereas no significant change was observed in p16 expression. Ang II = angiotensin II; Ad-CYR61 = adenoviral vectors expressing CYR61; Ad-AS-CYR61 = adenoviral vectors expressing antisense CYR61; Ad-GFP = adenoviral vector expressing green fluorescence proteins; CYR61 = cysteine-rich angiogenic protein 61; hCSMC = human coronary artery smooth muscle cell; ns = not significant. *p<0.01, †p<0.001.

• 

  Figure 6 p53 expression of Ang II-induced senescent hCSMCs was through ERK/p38 MAPK/CYR61 signaling pathway. (A) Western blot analysis shows that Ang II (100 nM, 240 minutes) induced the phosphorylation of ERK1/2 and p38 MAPK, which was inhibited by ARB, Fima (1 μM). (B) Ang II-induced CYR61 mRNA expression is mediated by both ERK1/2 and p38 MAPK. Scatter plot with bar shows data from 3 independent experiments. Values are given as mean±standard deviation (n=3). (C) Ang II-induced activation of ERK and p38 MAPK are inhibited by PD98059 (20 μM, 20 minutes) and SB203580 (10 μM, 20 minutes), respectively, but ERK1/2 was activated by SB203580. CYR61 expression is attenuated both by PD98059 and SB203580, whereas p53 expression levels are suppressed only by PD98059, whereas p16 expression level was decreased only by SB203580, respectively. Ang II = angiotensin II; ARB = Ang II type 1 receptor blocker; CYR61 = cysteine-rich angiogenic protein 61; Fima = fimasartan; hCSMC = human coronary artery smooth muscle cell. *p<0.01, †p<0.001.

• 

  Figure 7 The proposed signaling pathways of Ang II-induced hCSMCs senescence. ARB, Fima, may contribute to anti-senescence effects by inhibiting ERK/p38 MAPK/CYR61/p53 signaling pathway. Ang II = angiotensin II; ARB = Ang II type 1 receptor blocker; CYR61 = cysteine-rich angiogenic protein 61; Fima = fimasartan; hCSMC = human coronary artery smooth muscle cell.

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