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 and human atherosclerotic lesions [17
]. Mensink and colleagues [18
] demonstrated an increase in GJIC in VSMCs incubated with basic fibroblast growth factor and GJIC was a candidate for mediating the corresponding effect in vivo
. We recently reported that increased Cx43 expression following stimulation of VSMCs with Ang II and insulin-like growth factor-1 (IGF-1) contributed to more proliferation in the SMCs of saphenous vein than in the internal mammary artery [2
]. Gap junctions are the structural basis of intercellular communication between two adjacent cells and serves as channel for direct intercellular exchange of ions, small molecular metabolites, and as secondary messengers [19
]. In most VSMCs, Cx43 expression and function seem dominant [20
]. However, the regulation of Cx43 expression is not clear. In the present study, we investigated the effect of Ang II on the Cx43 expression and function and found that Cx43 was involved in the proliferation and migration of hSV SMCs. In addition, we provide new evidence that the Ang II-dependent increase in Cx43 expression is regulated by transcription factor, AP-1, via the ERK ½, p38 MAP kinases and JNK.
Over the past several years, a number of observations have suggested that Ang II plays an important role in various cardiovascular disease associated with VSMC proliferation such as hypertension, atherosclerosis, stenosis following balloon angioplasty and restenosis post-CABG [2
]. Recent studies revealed that the upregulation of Cx43 may relate to Ang II [22
]. In our study, we demonstrate that Ang II induces increased protein expression of Cx43 in the hSV SMCs in a dose- and time-dependent manner via AT1
receptor. The increased expression of Cx43 may result in the increase in GJIC in VSMCs. Ang II is a multifunctional peptide that has numerous actions on VSMCs including proliferation [2
] and regulation of Cx43 expression [24
]. In cardiac cells, Ang II induced activation of ERK1/2 and p38 pathways and is thought to play an important role in the upregulation of gap junctions under patho-physiological conditions [25
]. In rat hepatic epithelial cells, Ang II upregulates the expression of Cx43, which subsequently results in the changes of cellular metabolic conditions and secondary messengers [26
]. In atherosclerotic aorta of rabbits, statins reduce Cx40 and Cx43 expression by inhibition of angiotensin receptor [27
]. We have recently reported that under ex vivo
conditions connexin43 expression in both saphenous vein and internal mammary artery was influenced by angiotensin II and IGF-1 and may contribute to the pathogenesis of vein graft disease following coronary artery bypass grafting [2
]. These results correlate with the findings in the literature that under in vitro conditions, angiotensin II can increase gap junctions and intercellular communication in cardiac cells [14
] and connexin43 expression in WB rat liver epithelial cells [24
]. Increased connexin43 expression in human saphenous veins in culture is also associated with intimal hyperplasia [1
]. Thus, the regulation of Ang II-induced Cx43 expression in hSV SMCs may provide an explanation or elucidate the cause for the selective failure of hSV bypass grafts due to restenosis following CABG.
In our study, we found that the transfection of Cx43 alone was able to induce the proliferation of hSV SMCs. Conversely, silencing of Cx43 expression was able to inhibit the proliferation and migration induced by Ang II. These results are consistent with previous reports to show the participation of Cx43 in cell growth, differentiation and the development of intimal hyperplasia [1
]. Also, Christos et al.
also found that with a reduction in Cx43 expression of hypercholesterolemic mice, there is limited neointima formation after acute vascular injury accompanied by a decrease in the inflammatory response, and reduced VSMC migration and proliferation [28
]. Thus, Cx43-mediated GJIC between VSMCs may play a role in the proliferative and migratory responses. The underlying mechanisms are unclear. The findings in our previous study have shown that cyclin E play an important role in the proliferation of VSMCs in human atherosclerotic plaque [3
]. In this study, we found that cyclin E was involved in the Cx43-induced proliferation of VSMC after Ang II stimulation. Cx43 may also regulate cell cycle inhibitor proteins via p27 and p21, since the upregulation of Cx43 accompanies an increase in p27 and p21 expression levels in cancer cells [29
]. These results provide insight into the direct correlation of Cx43 expression and cell cycle proteins. In our study we have not examined the relationship between Cx43 expression and p27 and p21 proteins in VSMCs. However, increased expression and activity of Cx43 in VSMCs increased proliferation, which is different from the studies in cancer cells [29
]. None-the-less, Gap junction channels provide an enclosed conduit for direct exchanges of signaling molecules, including ions and small metabolites, between cells [1
]. It is possible that this system of communication allows cells to review the functional state of their neighbors and regulate MAP kinases in a feedback manner to induce cell proliferation.
The mechanisms underlying migration and proliferation in VSMCs are different. The processes of migration include remodeling of the cytoskeleton, changing the adhesiveness of the cell to the matrix, and activating motor proteins. The key intracellular molecules that are involved in the migration process include phosphatidylinositol 3-kinase, calcium-dependent protein kinase, Rho-activated protein kinase, p21-activated protein kinases, LIM kinase, and mitogen-activated protein kinases [30
]. However, the proliferation of VSMCs is due to increase in cell number associated with cell cycle proteins, DNA synthesis, and mitosis of cells, may involve some of the same enzymes as in the migration process [31
]. There are some reports in the literature demonstrating that increase in Cx43 expression induces migratory and proliferative activity in H9c2 cells and proepicardial cells [32
]. Cx43 might be one of the downstream target genes regulated by Wnt-3a [33
]. Obviously, further studies are warranted to elucidate and dissect cellular and molecular mechanisms underlying Cx43-induced proliferation and migration of VSMCs.
JNK and MAP kinase have been reported to be important intracellular signaling pathways that regulate Cx43 expression [34
]. However, many of the signaling events relevant to cell proliferation and differentiation are mediated through activation of transcription factors via MAP kinases, which can be upregulated by Ang II [36
]. Activation of the ERK, p38 and JNK signaling pathways may participate in the regulation of cardiac gap junctions [25
]. Here, our results confirm that Ang II can modulate the expression of Cx43 via the ERK1/2, p38 MAPK and JNK in hSV SMCs. However, the precise role of MAPKs in the regulation of Cx43 expression and activity in VSMCs is uncertain.
We hypothesized that Ang II mediates its effect through the ERK1/2, P38 and JNK pathways via the activation of transcription factor AP-1. AP -1 is part of a family of dimeric transcription factors comprised of either c-Jun homodimers or heterodimers of jun/fos family members [38
]. Dominant-negative AP-1 (A-Fos) has been proven to inhibit AP-1 activity in epithelial tumor cell [39
] and cardiac cells [40
]. In cultured neonatal rat cardiomyocytes, amphetamine-activated Cx43 gene expression was through the JNK and AP-1 pathways [37
]. The AP-1 and its promoter site are likely involved in the cytokine regulation of Cx43 [41
]. Activation of ERK1/2 induces overexpression of c-fos mRNA and enhanced AP-1 DNA-binding activity [42
]. Meanwhile, activation of p38 MAPK also results in phosphorylation of c-Jun and ATF-2 transcription factors, increasing their trans
-activating AP-1 activity [43
]. In our study, Ang II induced Cx43 expression via AP-1 transcriptional activity in hSV SMCs.
In summary, we have delineated cellular pathways to further understand the molecular mechanisms involved in the role of Cx43 in the proliferation and migration in hSV SMCs. These findings may explain, at least in part, the proliferative vasculopathy observed in vein graft disease. Further understanding of the expression of Cx43 in human venous bypass vessels has great clinical significance and could provide the basis for new approaches to better and effective treatment and to contain the morbidity and mortality associated with vein graft disease.