Korean J Physiol Pharmacol.  2015 Nov;19(6):499-506. 10.4196/kjpp.2015.19.6.499.

Pitavastatin Regulates Ang II Induced Proliferation and Migration via IGFBP-5 in VSMC

• Ha YM
• Nam JO
• Kang YJ

• KMID: 2070789
• DOI: http://doi.org/10.4196/kjpp.2015.19.6.499

Abstract

Angiotensin II (Ang II), a key mediator of hypertensive, causes structural changes in the arteries (vascular remodeling), which involve alterations in cell growth, vascular smooth muscle cell (VSMC) hypertrophy. Ang II promotes fibrotic factor like IGFBP5, which mediates the profibrotic effects of Ang II in the heart and kidneys, lung and so on. The purpose of this study was to identify the signaling pathway of IGFBP5 on cell proliferation and migration of Ang II-stimulated VSMC. We have been interested in Ang II-induced IGFBP5 and were curious to determine whether a Pitavastatin would ameliorate the effects. Herein, we investigated the question of whether Ang II induced the levels of IGFBP5 protein followed by proliferation and migration in VSMC. Pretreatment with the specific Angiotensin receptor type 1 (AT1) inhibitor (Losartan), Angiotensin receptor type 2 (AT2) inhibitor (PD123319), MAPK inhibitor (U0126), ERK1/2 inhibitor (PD98059), P38 inhibitor (SB600125) and PI3K inhibitor (LY294002) resulted in significantly inhibited IGFBP5 production, proliferation, and migration in Ang II-stimulated VSMC. In addition, IGFBP5 knockdown resulted in modulation of Ang II induced proliferation and migration via IGFBP5 induction. In addition, Pitavastatin modulated Ang II induced proliferation and migration in VSMC. Taken together, our results indicated that Ang II induces IGFBP5 through AT1, ERK1/2, P38, and PI3K signaling pathways, which were inhibited by Pitavastatin. These findings may suggest that Pitavastatin has an effect on vascular disease including hypertension.

Keyword

Hypertension; IGFBP-5; Pitavastatin; Proliferation; VSMC

MeSH Terms

Angiotensin II
Angiotensins
Arteries
Cell Proliferation
Heart
Hypertension
Hypertrophy
Insulin-Like Growth Factor Binding Protein 5*
Kidney
Lung
Muscle, Smooth, Vascular
Vascular Diseases
Angiotensin II
Angiotensins
Insulin-Like Growth Factor Binding Protein 5

Figure

• 

  Fig. 1 IGFBP-5 expression is induced by Ang II in VSMC. Cells were treated with Ang II 0.1 µM, 0.5 µM, 1 µM for 12 hrs (A). Cells were treated with Ang II 0.5 µM for 0, 1, 3, 6, 12, 24 hrs (B). After treatment, detection of protein by Western blot analysis. These data are representative of three experiments. *p<0.05 **p<0.001 compared with time 0.

• 

  Fig. 2 Pitavastatin reduced phosphorylation of MAPK and expression of Egr1 induced in Ang II-stimulated A7r5. Cells were treated with Ang II 0.5 µM. VSMC were incubated with Ang II for 0, 5, 10, 15, 30, 60, 90 min respectively (A). Cells were treated with Pitavastatin for 30 min before Ang II 0.5 µM for 15 min (B). p-JNK, p-ERK1/2, p-p38, p-Akt and Egr1 were determined by western blot analysis. Cells were transfected with ERK1/2 siRNA and then treated with Ang II 0.5µM for 12 hrs (C). Cells were transfected with Egr1 siRNA and then treated with Ang II 0.5µM for 12 hours (D). After treatment, IGFBP5 protein expression was measured by Western blot. These data are representative of three experiments.

• 

  Fig. 3 Expression of IGFBP5 is induced by AT1. Cells were pretreated with Losartan (1 µM) and PD123319 (1 µM) for 1 hr and then incubated with Ang II 0.5 µM for 12 hrs. Protein level was determined by Western blot (A). And treatment with Ang II 0.5 µM for 24 hrs, cell proliferation was determined by MTT assay (B). Transfection with AT1 siRNA or control siRNA and IGFBP5 recombinant treatment and then treated with Ang II 0.5 µM for 12 hrs. Protein level was determined by Western blot (C). Treatment with Ang II 0.5 µM for 24 hrs, cell proliferation was determined by MTT assay (D). These data are representative of three experiments. *p<0.001 compared with untreated cells, #p <0.001 compared with Ang II.

• 

  Fig. 4 Effects of inhibiting MAPKs on Ang II-induced IGFBP5 expression in VSMC. Cells were pretreated with U0126 (1 µM) and PD98059 (1 µM) for 1 hr and then incubated with Ang II 0.5 µM for 12 hrs. Protein level was determined by western blot (A). Cells were pretreated with SB-203580 (10 µM), SP600125 (25 µM), and LY294002 (10 µM) for 1hr and then incubated with Ang II 0.5 µM for 12 hrs. Protein level was determined by western blot (B, C). Cells were pretreated with U0126, PD98059, SB203580, SP600125, and LY294002 for 1 hr and then incubated with Ang II 0.5 µM for 24 hrs. Cell proliferation was determined by MTT assay (D). These data are representative of three experiments. *p<0.001 compared with untreated cells, #p<0.001 compared with Ang II.

• 

  Fig. 5 Effects of IGFBP5 on Ang IIinduced proliferation and migration in VSMC. Cells were transfected with IGFBP5 siRNA and then treated with Ang II 0.5 µM for 12 hrs. IGFBP5 protein expression was measured by western blot (A). Cells were treated with Ang II 0.5 µM for 24 hrs. Cell proliferation was measured by MTT assay (B) Cell migration was measured by scratch wound assay (C, D). These data are representative of three experiments. *p<0.001 compared with untreated cells, #p<0.001 compared with Ang II.

• 

  Fig. 6 Pitavastatin modulates Ang IIinduced IGFBP5 expression, proliferation and migration in VSMC via IGFBP5 expression. Cells were pretreated with Pitavastatin 1.0 µM for 1hr and then incubated with Ang II 0.5 µM for 12 hrs. IGFBP5 protein expression was measured by western blot (A). And cells were treated with Ang II 0.5 µM for 24 hrs. Cell proliferation was measured by MTT assay (B). Cell migration was measured by scratch wound assay (C, D). These data are representative of three experiments. *p<0.001 compared with untreated cells, #p<0.001 compared with Ang II, $p<0.005 compared with untreated cells.

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