The role of runt-related transcription factor 2 in arterial smooth muscle cell mineralization.

摘要

Medial arterial calcification is the deposition of calcium phosphate mineral in the medial layer of the vessel. It frequently manifests in patients with type II diabetes mellitus and end stage renal disease, where it is known to contribute to the morbidity and mortality of these patients. It is hypothesized that the mechanisms responsible for regulating arterial calcification are the same as those that regulate bone formation and bone remodeling. In fact, several bone proteins such as, type I collagen, bone sialoprotein, alkaline phosphatase, osteocalcin, osteopontin, and runt-related transcription factor 2 (Runx2), have been associated with medial calcification both in vivo and in vitro. The presence of the transcription factor Runx2 in arterial medial cells undergoing mineralization is of particular interest because Runx2 is required for osteoblast differentiation and osteoblasts are the bone-forming cells that synthesize and mineralize extracellular matrix. In this dissertation experiments examining the role of Runx2 in bovine arterial smooth muscle cell mineralization are described. The development of an in vitro arterial calcification model in which bovine aortic smooth muscle cells (BASMCs) are treated with elevated inorganic phosphate (Pi) and subsequently analyzed for extracellular matrix mineralization and changes in gene expression is described. Using this model, I show that Pi-treated BASMCs up-regulate Runx2 as part of a phenotypic transition that occurs concomitant with mineral deposition. To investigate the role of Runx2 in Pi-induced BASMC mineralization, I generated stable cell lines, which constitutively express a dominant negative Runx protein (DNRunx) that inhibits Runx regulation of downstream targets. I show that DNRunx BASMCs deposit more extracellular calcium than vector control BASMCs (VC BASMCs) when treated with elevated Pi and they have reduced levels of osteopontin and increased levels of osterix expression. Finally, I describe the characterization of BASMC cells constitutively expressing wild type Runx2 (WTRunx2) from a retroviral promoter. Elucidation of the role of Runx2 in Pi-induced BASMC mineralization may contribute to a greater understanding of the mechanisms regulating medial arterial calcification.