Involvement of connexin 43 in angiotensin II-induced migration and proliferation of saphenous vein smooth muscle cells via the MAPK-AP-1 signaling pathway

Abstract

Proliferation and migration of vascular smooth muscle cells (VSMCs) lead to intimal thickening and influence the long-term patency of venous graft post coronary arterial bypass graft. There is increasing evidence that connexins are involved in the development of intimal hyperplasia and restenosis. We assessed connexin 43 (Cx43) expression and its role in angiotensin II-induced proliferation and migration of smooth muscle cells and the signal pathways involved in human saphenous vein bypass conduits. Angiotensin II significantly increased gap junctional intercellular communication and induced the expression of Cx43 in human saphenous vein SMCs in a dose- and time-dependent manner through angiotensin II type 1 receptor. The effect of angiotensin II was blocked by siRNA of ERK 1/2, p38 MAPK and JNK, respectively. Overexpression of Cx43 markedly increased the proliferation of saphenous vein SMCs. However, siRNA for Cx43 inhibited angiotensin II-induced proliferation, cyclin E expression and migration of human saphenous vein SMCs. In dual-luciferase reporter assay, angiotensin II markedly activated AP-1 transcription factor, which was significantly attenuated by a dominant-negative AP-1 (A-Fos) with subsequent inhibition of angiotensin II-induced transcriptional expression of Cx43. These data demonstrate the role of Cx43 in the proliferation and migration of human saphenous vein SMCs and angiotensin II-induced Cx43 expression via mitogen-activated protein kinases (MAPK)-AP-1 signaling pathway.

Fig. 1

Ang II induced Cx43 protein expression through AT₁ receptor in VSMCs. (A) Ang II (10⁻⁷ mol/L) induced Cx43 protein expression in SV SMCs in a time-dependent manner. (B) Ang II induced Cx43 protein expression of SV SMCs at 1 h in a dose-dependent manner. The relative amount of Cx43 protein expression was measured by Western blot. (C) Ang II receptor antagonists, losartan (10⁻⁶ mol/L, AT₁ receptor-specific) and PD-123319 (10⁻⁶ mol/L, AT₂ receptor-specific), were added to the cultured medium for 30 minutes followed by stimulation of the cells with 10⁻⁷ mol/L Ang II for an additional 24 hours. The relative amount of Cx43 protein expression was measured by Western blot. Each bar represents the ratio of Cx43/GAPDH (mean ± SE) from three independent experiments. *P<0.05 compared with control group, §P<0.01 compared with control group, #P<0.01 compared with Ang II group.

Fig. 2

Ang II increased gap junctional intercellular communication in saphenous vein smooth muscle cells. SV SMCs were treated with Ang II (10⁻⁷ mol/L) for 1 hour, and gap junctional intercellular communication capacity was assayed by the Scrape Loading/Dye Transfer method. The number of communicating cells in the untreated and treated cultures was counted under a light microscope (magnification 10×). GJIC was expressed as percentage of the control. The bar represents percentage of the control (mean ± SE) for three independent experiments.

Fig. 3

Effect of Cx43 siRNA and overexpression of pcDNA^™3.1- Cx43 on the Cx43 expression in saphenous vein smooth muscle cells. (A) VSMCs were transfected with control-siRNA or empty plasmid or pcDNA^™3.1- Cx43 or Cx43-siRNA for 48 h. (B) SV SMCs were transfected with siRNA against Cx43 and non-silencing siRNA and total protein expression of Cx43 was determined by Western blot analysis after 48h and 72h. The cells which were transfected with Cx43-siRNA were treated with Ang II (10⁻⁷ mol/L) for 24 h, lysed and protein expression of Cx43 was measured. Values are mean ± SE for three independent experiments. §P<0.01 compared with control group. # P<0.01 compared with Ang II+Cx-siRNA group.

Fig. 4

Effect of Cx43 overexpression on the proliferation of VSMCs. After transfection with pcDNA^™3.1- Cx43 and empty vector, VSMCs were treated with Ang II (10⁻⁷ mol/L) for 24 h. (A) BrdU incorporation into VSMC was increased by Cx43 overexpression or Ang II stimulation. (B) Cell number of VSMC was significantly increased by Cx43 overexpression or Ang II stimulation. Values are mean± SE for three independent experiments. §P<0.01 compared with control group.

Fig. 5

Effect of Cx43–siRNA on Ang II-induced-proliferation of VSMCs and expression of cyclin E protein. After transfection with Cx43-siRNA and control-siRNA, VSMCs were treated with Ang II (10⁻⁷ mol/L) for 24 h. (A) BrdU incorporation into VSMC was inhibited by transfection with Cx43-siRNA. (B) Cell number of VSMC was significantly decreased by transfection with Cx43-siRNA. (C) Expression of cyclin E was inhibited by transfection of the cells with Cx43-siRNA. Values are mean± SE for three independent experiments.

Fig. 6

Effect of Cx43–siRNA on the migration of VSMCs. After transfection with Cx43-siRNA and control-siRNA, VSMCs were treated with Ang II (10⁻⁷ mol/L) for 24 h and subsequently studied cells migration by using transwell apparatus. Photograph of migrated SMC of saphenous vein were shown under microscope (magnification 10×). Each bar represents mean ± SE of cell migrated per high-power microscope for three independent experiments.

Fig. 7

Effect of siRNA for ERK1/2, p38 and JNK on Ang II -induced expression of Cx43 in SMCs of saphenous vein. (A) hSV SMCs were stimulated with Ang II (10⁻⁷ mol/L) for 10 min (ERK and p38) and 30 min (JNK). The cells were lysed and protein expression of p-ERK, p-p38 and p-c-jun was examined. (B) After transfection of the cells with siRNA for ERK 1/2, p38, and c-jun and control-siRNA, VSMCs were treated with Ang II (10⁻⁷ mol/L) for 24 h. Proteins were quantified and each well was loaded with 20 μg of protein. Each bar represents the ratio of Cx43/GAPDH (mean ± SE) from three independent experiments. §P<0.01 compared with control group. # P<0.01 compared with Ang II group.

Fig. 8

Involvement of activation of transcription factor AP-1 in Ang II induced connexin43 expression in saphenous vein SMCs. (A) VSMCs were co-transfected with pAP-1-Luc and 0.2 μg of Renilla luciferase reporter control vector in the absence or presence of dominant-negative AP-1 (A-Fos). Twenty-four hours after transfection, the cells were treated with Ang II (10⁻⁷ mol/L) for 3 hours, the cells were lysed and luciferase activities were measured. VSMCs were transfected with either control empty plasmid or dominant-negative AP-1 (A-Fos). After overnight recovery, cells were starved for 24 h and then either left untreated or treated with Ang II (10⁻⁷ mol/L) for 24 hours. (B) The cells were lysed and Cx43 protein was measured. Data are mean ± SE of five observations. §P<0.01 compared with control group. #P<0.01 compared with Ang II group.