Original Article
Receptor activator of nuclear factor κB ligand and osteoprotegerin regulate aortic valve calcification

https://doi.org/10.1016/j.yjmcc.2003.09.015Get rights and content

Abstract

Objective. – Recent studies have suggested that valvular calcification in calcific aortic stenosis (AS) may be actively regulated. “Receptor Activator of Nuclear factor κB Ligand” (RANKL) and osteoprotegerin (OPG) are members of a cytokine system involved in bone turnover and vascular calcification. Their role in calcific AS is not known.

Methods and Results. – By immunohistochemistry using human aortic valves, RANKL was not expressed at relevant levels in controls but detectable in AS. OPG expression was marked in controls but significantly lower in AS. Areas containing focal calcification exhibited significantly less OPG-positive cells as compared to non-calcified regions. Stimulation with RANKL lead to a significant rise in matrix calcification, nodule formation, alkaline phosphatase activity, expression of the bone-type isoenzyme of alkaline phosphatase, and expression of osteocalcin in cultured human aortic valve myofibroblasts. Moreover, RANKL increased DNA binding of the essential osteoblast transcription factor cbfa-1.

Conclusion. – RANKL and OPG are differentially expressed in calcific AS. In cultured human aortic valve myofibroblasts, RANKL promotes matrix calcification and induces the expression of osteoblast-associated genes, indicating a transition towards an osteogenic phenotype. These results suggest that the RANKL-OPG pathway may regulate valvular calcification in calcific AS?

Introduction

Non-rheumatic, calcific aortic valve stenosis (AS) is the most frequent heart valve disease in developed countries and the main cause of heart valve replacement in the elderly [1]. Recent studies indicate that calcific AS might be based on a chronic inflammatory process involving activated macrophages and T-lymphocytes [2], [3]. Also, newer reports suggest that aortic valve calcification could be an actively regulated process that involves mechanisms of bone development [4], [5], [6]. However, other authors have proposed that aortic valve calcification is a passive process of mineral deposition around a core of apoptotic cells, lipid droplets, and cellular debris [7].

Recently, a new cytokine pathway of the tumor necrosis factor superfamily has been discovered that is involved in the regulation of bone resorption and vascular calcification. This cytokine system consists of the transmembrane protein “receptor activator of nuclear factor κB” (RANK), its ligand (RANKL), and the soluble receptor osteoprotegerin (OPG). Mouse studies have shown that the RANKL–RANK pathway is the gatekeeper of osteoclast differentiation and activation [8], and that RANKL is a key regulator of osteoclastogenesis and lymphocyte development [9]. OPG is a soluble decoy receptor that binds to RANKL, thereby inhibiting the interaction of RANKL and RANK. OPG is expressed at high concentrations by a variety of tissues and cell types including arterial smooth muscle cells and endothelial cells, whereas RANKL and RANK are not expressed in vascular tissue under physiologic conditions [10], [11]. Deletion of the OPG gene leads to severe calcification of aorta and renal arteries, and to an expression of RANKL and RANK in the calcified areas [11], suggesting that RANKL might promote vascular calcification and that OPG may have a protective role [12]. The impact of RANKL and OPG on valvular calcification is unknown.

The present study, therefore, examined the expression of RANKL and OPG in human aortic valves with and without calcific AS, and assessed the influence of RANKL on calcification and the development of an osteogenic phenotype in cultured human aortic valve myofibroblasts (HAVMs).

Section snippets

Aortic valve tissue

Human tricuspid aortic valves were obtained from patients undergoing valve replacement for calcific AS. As controls, aortic valves were obtained at autopsy from patients without clinical and morphological AS. Informed consent was obtained, and the investigation protocol was approved by the institutional ethics committee. Valve leaflets were rinsed in cold saline to remove blood, frozen in liquid nitrogen and stored at –80 °C.

Tissue protein extraction

Native, untreated valve tissue from five stenotic and three control

OPG expression is inversely correlated with aortic valve calcification

The expression and localization of OPG were assessed by western blotting and immunohistochemistry. Western blotting of native protein extracts for OPG demonstrated bands at 40 kDa in control valves and no detectable bands in stenotic valves (Fig. 1A ). Immunohistochemistry showed a high proportion of OPG-positive cells in control valves (Fig. 1B). Staining of thickened, but not calcified, areas of stenotic valves demonstrated an increased total cell count but a reduced percentage of

Discussion

RANKL and OPG are part of a novel regulatory system of the tumor necrosis factor ligand and receptor family and are thought to be involved in the regulation of vascular calcification [11], [20]. In the present report, we demonstrate for the first time that RANKL and OPG are expressed in human aortic valves. By immunohistochemistry, marked expression of RANKL is noted in calcified valves while only scattered positive cells are shown in some of the controls. OPG is strongly expressed in normal

Acknowledgements

This study was supported in part by the Research Fund of the Faculty of Clinical Medicine Mannheim, University of Heidelberg, using the facilities of the Center of Medical Research, University Hospital Mannheim. The authors thank Ms. Claudia Liebetrau for expert laboratory assistance.

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    Presented in part as an abstract at the Annual Scientific Session of the American College of Cardiology, 2002.

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