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

TLDR

Vascular calcification is an ectopic calcification that commonly occurs in atherosclerosis. The study investigated whether tumor necrosis factor‑α (TNF‑α), a cytokine present in atherosclerotic lesions, promotes in‑vitro vascular calcification. The authors treated a subpopulation of bovine aortic smooth muscle cells capable of osteoblastic differentiation with TNF‑α and measured osteoblastic differentiation and mineralization. TNF‑α induced osteoblast‑like morphology, increased alkaline phosphatase activity and mRNA, enhanced matrix mineralization, and these effects were mediated through the cAMP pathway, as shown by PKA inhibition, elevated cAMP, and increased DNA binding of Osf2, AP1, and CREB.

Abstract

Background —Vascular calcification is an ectopic calcification that commonly occurs in atherosclerosis. Because tumor necrosis factor-α (TNF-α), a pleiotropic cytokine found in atherosclerotic lesions, is also a regulator of bone formation, we investigated the role of TNF-α 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-α, and osteoblastic differentiation and mineralization were assessed. Treatment of vascular cells with TNF-α 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-α 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-α. Consistent with this, the intracellular cAMP level was elevated after TNF-α treatment. Electrophoretic mobility shift assay demonstrated that TNF-α 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-α enhances in vitro vascular calcification by promoting osteoblastic differentiation of vascular cells through the cAMP pathway.