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

https://doi.org/10.1016/j.yjmcc.2008.03.002Get rights and content

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.

Introduction

Intimal hyperplasia is a proliferative vasculopathy that causes stenosis in the venous bypass graft, a pathophysiologic feature leading to occlusion of the vessel lumen [1], resulting in failure rates of 20% and 50% at 5years and 10years, respectively [2]. Proliferation and migration of vascular smooth muscle cells (VSMCs) into the sub-intimal space play an important role in intimal thickening in atherosclerosis and restenosis and influences the long-term patency of the venous graft [3]. Initiation and progression of the atherosclerotic plaque and restenotic vessel involve complex patterns of interaction between the cells of the arterial wall, in which cytokines, chemokines, and growth factors are known to play a critical role [4], [5]. For example, angiotensin II (Ang II), a potent growth factor, stimulates proliferation and migration of VSMCs, and induces the accumulation and deposition of collagen through Ang II type 1 (AT1) receptor; all factors contributing to the pathogenesis of atherosclerosis [6].

Another form of cell-to-cell interaction involves direct exchange of small cytosolic components via gap junctions, which are composed of connexon hemichannels, each of which are made of 6 connexin (Cx) molecules [7]. Three subtypes of Cx, Cx37, Cx40, and Cx43, are found in the cells of the vasculature, with Cx43 being the predominant one [7], [8]. Changes in connexin expression and function have been found in association with phenotypic changes in VSMCs during the progression of atherosclerotic lesions and following injury in large arteries [9]. Also, Ang II can activate the expression of Cx43 in the myocardial cells of rats in vitro[10]. However, little is known about the relationship between Ang II and Cx43 expression in VSMCs of the human saphenous vein (hSV). In this study, we examined the Cx43 expression and its role in the proliferation and migration of VSMCs. Further, we delineated the signaling pathways involved in the proliferation of smooth muscle cells in hSV bypass conduits following stimulation with Ang II.

Section snippets

Specimen collection, processing and culture of smooth muscle cell

The experimental protocol for this study was approved by the Institutional Review Board of Creighton University. The hSV specimens (mean patient age 71.5 ± 3.7years) left over from the bypass surgical procedure were used for the study. Specimens were collected fresh in ice-cold University of Wisconsin solution, which is used to maintain cellular and tissue integrity in the organs for transplantation [11]. The hSV conduits were immediately brought to the laboratory where all of the procedures

Ang II-induced Cx43 expression through AT1 receptors in the SMCs of human venous bypass vessels

Cx43 protein expression in hSV SMCs was evaluated in a time- and dose-dependent manner. After treatment with Ang II (10 7mol/L), the Cx43 expression was increased significantly at 0.5h, peaked at 1 h and remained at an elevated level even after 3h in the SV samples (Fig. 1A). Also, Ang II (10 7and 10 6mol/L) induced a significant maximal response in SV at 1 h in a dose-dependent manner (Fig. 1B).

To determine which AT receptor subtype is involved in Ang II-induced Cx43 protein expression in hSV

Discussion

The hSV is the most commonly employed CABG conduit. Unfortunately, intimal hyperplasia leading to stenosis and occlusion of hSV commonly limit long-term results following coronary artery bypass graft (CABG) surgery [15]. The mechanisms involved in the intimal hyperplasia are proliferation and migration of medial smooth muscle cells towards the sub-intimal space [16]. There is increasing evidence that connexins are involved in the development of intimal hyperplasia and restenosis involving mouse

Acknowledgments

This study was supported by National Institutes of Health grants R01HL070885 (D.K.A.) and R01HL073349 (D.K.A.), and the State of Nebraska Tobacco Settlement Funds (D.K.A.). We thank Nebraska Heart Institute for providing us the venous bypass specimens.

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