Cross-talk between angiotensin II and IGF-1-induced connexin 43 expression in human saphenous vein smooth muscle cells

Abstract

Vascular restenosis following coronary artery bypass graft can cause major clinical complications due to intimal hyperplasia in venous conduits. However, the precise underlying mechanisms of intimal hyperplasia are still unclear. We have recently reported that increased expression of connexin43 (Cx43) is involved in the proliferation of vascular smooth muscle cells (SMCs) in human saphenous vein (SV). In this study, we investigated the signalling transduction pathway involved in Cx43 expression and SV SMC proliferation. Angiotensin-II (AT-II, 100 ng/ml) increased AT-II receptor 1 (AT-1R) protein expression and insulin-like growth factor-1 (IGF-1) (100 ng/ml) up-regulated IGF-1 receptor (IGF-1R) protein expression in SV SMCs. Interestingly, AT-1R expression was also increased by IGF-1 treatment, and IGF-1R expression was increased by AT-II treatment, which was blocked by siRNA-IGF-1R and siRNA-AT-1R, respectively. Furthermore, the effect of AT-II and IGF-1 signal cross-talk i nducing up-regulation of their reciprocal receptors was blocked by siRNA against extracellular signal-regulated kinases 1/2 (Erk 1/2) in SMCs of SV. Moreover, AT-II and IGF-1-induced Cx43 expression via phosphorylation of Erk 1/2 and activation of transcription factor activator protein 1 (AP-1) through their reciprocal receptors in SV SMCs. These data demonstrate a cross-talk between IGF-1R and AT-1R in AT-II and IGF-1-induced Cx43 expression in SV SMCs involving Erk 1/2 and downstream activation of the AP-1 transcription factor.

Fig 1

IGF-1 and AT-II induced the expression of IGF-1R and AT-1R in SV SMC. SV SMCs were treated with AT-II (10⁻⁷ mol/l) or IGF-1 (100 ng/ml) for 24 hrs, and the expression of IGF-1R and AT-1R protein was assayed by flow cytometry. The bar represents percentage of IGF-1R⁺ and AT-1R⁺ cells (mean ± S.E.) for three independent experiments. ^#P < 0.01 compared with control group. *P < 0.01 compared with IGF-1 group or AT II group.

Fig 2

Effect of AT-II and IGF-1 on the protein expression of AT-1R and IGF-1R in SV SMCs. (A) SMCs were transfected with siRNA against AT-1R and non-silencing siRNA, and total protein expression of AT-1R was determined by Western blot analysis after 48 and 72 hrs. (B) SMCs were transfected with siRNA against IGF-1R and non-silencing siRNA, and total protein expression of IGF-1R was determined by Western blot analysis after 48 and 72 hrs. (C) After transfection with IGF-1R-siRNA for 24 hrs, SMCs were untreated or treated with AT-II (10⁻⁷ mol/l) or IGF-1(100 ng/ml) for 24 hrs. (D) After transfection with AT-1R-siRNA for 24 hrs, SMCs were untreated or treated with AT-II (10⁻⁷ mol/l) or IGF-1 (100 ng/ml) for 24 hrs. The cells were lysed, and AT-1R and IGF-1R protein was measured. Data are mean ± S.E. of triplicate observations. ^#P < 0.01 compared with control group. *P < 0.01 compared with AT-II group. ^§P < 0.01 compared with IGF-1 group.

Fig 3

Effect of siRNA for Erk1/2 on AT-II and IGF-1-induced expression of their reciprocal receptor in SMCs of SV. (A) VSMCs were stimulated with AT-II (10⁻⁷ mol/l) or IGF-1 (100ng/ml) for 10 min. The cells were lysed and p-ERK was measured. (B) SMCs were transfected with siRNA against Erk 1/2 and non-silencing siRNA, and total protein expression of Erk 1/2 was determined by Western blot analysis after 48 and 72 hrs. (C) After transfection with Erk 1/2-siRNA, SMCs were treated with IGF-1 (100 ng/ml) for 24 hrs. (D) After transfection with Erk 1/2-siRNA, SMCs were treated with AT-II (10⁻⁷ mol/l) for 24 hrs. Proteins were quantified, and each well was loaded with 20 μg of protein. Data are mean ± S.E. of triplicate observations. ^#P < 0.01 compared with control group. *P < 0.01 compared with AT-II group or IGF-1 group.

Fig 4

Effect of siRNA for AT-1R, IGF-1R and Erk 1/2 on AT-II and IGF-1-induced expression of Cx43 (molecular mass of 43 kD) in SMCs of SV. (A) After transfection with AT-1R-siRNA and IGF-1R-siRNA, SMCs were treated with AT-II (10⁻⁷ mol/l) and IGF-1 (100 ng/ml) for 24 hrs. (B) After transfection with Erk-siRNA, SMCs were treated with AT-II (10⁻⁷ mol/l) and IGF-1 (100 ng/ml) for 24 hrs. Proteins were quantified, and each well was loaded with 20 μg of protein. Each bar represents the ratio of Cx43/GAPDH (mean ± S.E.) from three independent experiments. ^#P < 0.01 compared with control group. *P < 0.05 compared with control group. ^§P < 0.01 compared with AT-II group. ^&P < 0.01 compared with IGF-1 group.

Fig 5

Activation of transcription factor AP-1 in AT-II and IGF-1-induced Cx43 expression (molecular mass of 43 kD) in SV SMCs. (A) SMCs were co-transfected with pAP-1-Luc and 0.2 μg of Renilla luciferase reporter control vector in the absence or presence of siRNA against AT-1R, IGF-1R and Erk 1/2. Twenty-four hours after transfection, the cells were treated with AT-II (10⁻⁷ mol/l) or IGF-1(100 ng/ml) for 3 hrs. The cells were then lysed, and luciferase activities were measured. (B) SMCs were transfected with either control empty plasmid or A-Fos. After overnight recovery, cells were starved for 24 hrs, and then either left untreated or treated with AT-II (10⁻⁷ mol/l) or IGF-1(100 ng/ml) for 24 hrs. The cells were lysed and Cx43 protein was measured. Data are mean ± S.E. of five observations. In (A): ^#P < 0.01 compared with control group. *P < 0.01 compared with AT-II group. ^§P < 0.01 compared with IGF-1 group. In (B): ^#P < 0.01 compared with control group. ^&P < 0.05 compared with control group. *P < 0.01 compared with AT-II group. ^§P < 0.01 compared with IGF-1 group.