Cyclic AMP inhibits production of interleukin-6 and migration in human vascular smooth muscle cells

doi:10.1016/S0022-4804(02)00038-0

Journal of Surgical Research

Volume 109, Issue 1, January 2003, Pages 57-61

https://doi.org/10.1016/S0022-4804(02)00038-0 Get rights and content

Abstract

Background. Gene expression induced by tumor necrosis factor-α (TNF-α) is involved in the regulation of vascular smooth muscle cell (VSMC) proliferation and migration, two events critical to formation of stenotic vascular lesions. In some systems, elevating adenosine 3′,5′-cyclic monophosphate (cyclic AMP) inhibits TNF-α induced gene transcription. We recently demonstrated that interleukin-6 (IL-6) was chemotactic to VSMC. Therefore, we tested the hypothesis that elevating cyclic AMP would inhibit TNF-α-mediated IL-6 expression and VSMC migration.

Materials and methods. VSMC were cultured from saphenous vein remaining after coronary artery bypass grafting. Migration of VSMC through a porous membrane was determined. Intracellular cyclic AMP was elevated by exposing the cells to forskolin or 8-Br-cyclic AMP and was measured by radioimmunoassay. IL-6 was measured by enzyme-linked immunosorbent assay.

Results. TNF-α induced migration of VSMC in a concentration-dependent manner. Incubation of cells with forskolin significantly increased cyclic AMP. Co-incubation of cells with TNF-α in combination with 8-Br-cyclic AMP or forskolin inhibited migration by approximately 25 and 70%, respectively. Incubation with TNF-α increased release of IL-6 from VSMC 18-fold over basal. This stimulated release was inhibited by either 8-Br-cyclic AMP or forskolin. In cells stimulated with TNF-α, addition of an antibody to IL-6 reduced migration by 25%.

Conclusions. These data show that IL-6 produced by VSMC contributes to cell migration induced by TNF-α. Further, elevating cyclic AMP inhibited TNF-α-induced release of IL-6, and migration of VSMC. These results are consistent with the notion that mechanisms that increase intracellular cyclic AMP, such as activation of β-adrenergic receptors on VSMC, act as a brake on cell migration.

Introduction

Vascular intimal hyperplasia is a major component of cardiovascular disease. It is associated with atherosclerosis and formation of neointima after vascular injury such as occurs following balloon angioplasty. The resulting narrowing of the vessel lumen is the principal cause of grafted vessel closure and of chronic organ graft failure in general. Stenosis of the affected vessel involves vascular smooth muscle cell (VSMC) proliferation and migration, two processes which can occur together or independently of each other. Understanding the cellular signaling mechanisms that drive proliferation and/or migration is essential to developing appropriate therapy to control intimal hyperplasia.

The inflammatory cytokine, tumor necrosis factor-α (TNF-α), is expressed by VSMC in human atherosclerotic vessels and in VSMC of the neointima of vessels subjected to balloon injury but not in the smooth muscle cells of normal blood vessels 1, 2. In vivo experiments in genetically engineered mice unable to produce TNF-α showed that neointima formation in carotid arteries subjected to low shear stress was almost completely blocked [3]. When these mice were bred to express TNF-α on the surface of the smooth muscle cells, neointima formation and carotid stenosis were restored. Further, development of neointima in the coronary arteries of transplanted rabbit hearts was inhibited by treatment with the soluble receptor for TNF-α [4]. Such in vivo experiments, as the two cited above, provide strong evidence of a key role of this cytokine in the formation of the neointima. Additionally, TNF-α induces expression of a number of genes thought to be involved in regulating migration and proliferation of VSMC, including genes encoding interleukin-6 (IL-6) and IL-8 and the matrix degrading metalloproteinases (MMPs) 5, 6, 7. Adenosine 3′5′-cyclic monophosphate (cyclic AMP) acting through a cyclic AMP responsive element has been shown to regulate expression of a number of target genes [8]. We recently found that elevation of intracellular cyclic AMP inhibited myocardial cytokine gene expression, including IL-6, induced by either bacterial lipopolysaccharide or reactive oxygen 9, 10. Therefore, we tested the hypothesis that elevated cyclic AMP would inhibit migration of VSMC by suppressing production of chemotactic agents such as IL-6.

Section snippets

Cell culture

With IRB approval and patient consent, segments of saphenous vein unused in coronary bypass were stripped of adventitia and cut open, and the endothelium was removed by gentle scraping. Explants (2 × 2 mm) were placed luminal side down in a Petri dish and incubated with Dulbecco’s modified Eagle’s medium (DMEM) plus 20% fetal bovine serum (FBS) and antibiotics (penicillin, 100 IU/ml, and streptomycin, 100 μg/ml) at 37°C in room air plus 5% CO₂. After 1 to 2 weeks the tissue was removed and the

Results

Figure 1 shows the concentration response of TNF-α on migration of VSMC. There was increased migration induced by the cytokine up to 17-fold over control at 10 ng/ml. A concentration of 100 ng/ml significantly reduced migration compared to 10 ng/ml but was still greater then control. In all the following experiments a concentration of 10 ng/ml was used.

Figure 2 shows the effect of forskolin and 8-Br-cyclic AMP on migration induced by TNF-α. The addition of either significantly reduced

Discussion

Saphenous vein is used to bypass atherosclerotic lesions in both coronary and peripheral vessels. Long term, this grafting procedure is less than successful in that 50% of these grafted vessels will be occluded in 10 years [13]. The principal cause of stenosis and failure of the graft is neointimal hyperplasia, where smooth muscle cell proliferation and migration are major contributors [14]. In this study we investigated the effect of a major inflammatory cytokine expressed by smooth muscle

Acknowledgements

The authors thank Benjamin P. Mitchell for technical assistance. This work was supported by grants from the MedCen Foundation, the Clinical Research Center of the Medical Center of Central Georgia, and the American Heart Association, Mid-Atlantic Affiliate.

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Citation Excerpt :
This is important since the elevation in the local IL-6 expression might increase neointimal hyperplasia by increasing VSMC migration and proliferation. This point is supported by others who suggesting that IL-6 produced by VSMC contributes to cell migration and to neointima formation (Newman et al., 2003). It is important to note that these cells change also their morphological structure and they have synthetic phenotype as shown by TEM analysis.
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Cyclic AMP inhibits production of interleukin-6 and migration in human vascular smooth muscle cells

Abstract

Introduction

Section snippets

Cell culture

Results

Discussion

Acknowledgements

FEBS Lett.

Blood

Ann. Thorac. Surg.

Exp. Cell Res.

J. Biol. Chem.

Tumor necrosis factor-alpha in human arterial wall with atherosclerosis

Atherosclerosis

Proliferating arterial smooth muscle cells after balloon injury express TNF-α but not Interleukin-1 or basic fibroblast growth factor

Arterioscler. Thromb. Vasc. Biol.

Direct evidence for cytokine involvement in neointimal hyperplasia

Circulation

In vivo blockade of tumor necrosis factor-alpha in cholesterol-fed rabbits after cardiac transplant inhibits acute coronary artery neointimal formation

Circulation

Enhanced expression of vascular matrix metalloproteinases induced in vitro by cytokines and in regions of human atherosclerotic lesions

Ann. NY Acad. Sci.

Cytokine-stimulated human vascular smooth muscle cells synthesize a complement of enzymes required for extracellular matrix digestion

Circ. Res.