Tumor Necrosis Factor-a Promotes In Vitro Calcification of Vascular Cells via the cAMP Pathway

Background—Vascular calcification is an ectopic calcification that commonly occurs in atherosclerosis. Because tumor necrosis factor-a (TNF-a), a pleiotropic cytokine found in atherosclerotic lesions, is also a regulator of bone formation, we investigated the role of TNF-a in in vitro vascular calcification. Methods and Results—A cloned subpopulation of bovine aortic smooth muscle cells previously shown capable of osteoblastic differentiation was treated with TNF-a, and osteoblastic differentiation and mineralization were assessed. Treatment of vascular cells with TNF-a for 3 days induced an osteoblast-like morphology. It also enhanced both activity and mRNA expression of alkaline phosphatase, an early marker of osteoblastic differentiation. Continuous treatment with TNF-a for 10 days enhanced matrix mineralization as measured by radiolabeled calcium incorporation in the matrix. Pretreatment of cells with a protein kinase A–specific inhibitor, KT5720, attenuated cell morphology, the alkaline phosphatase activity, and mineralization induced by TNF-a. Consistent with this, the intracellular cAMP level was elevated after TNF-a treatment. Electrophoretic mobility shift assay demonstrated that TNF-a enhanced DNA binding of osteoblast specific factor (Osf2), AP1, and CREB, transcription factors that are important for osteoblastic differentiation. Conclusions—These results suggest that TNF-a enhances in vitro vascular calcification by promoting osteoblastic differentiation of vascular cells through the cAMP pathway. (Circulation. 2000;102:2636-2642.)