Wnt5a is associated with the differentiation of bone marrow mesenchymal stem cells in vascular calcification by connecting with different receptors

Abstract

Vascular calcification significantly affects the health of the elderly. Increasing evidence proved that vascular calcification is an actively regulated osteogenic process. The osteochondrocytic differentiation of mesenchymal stem cells (MSCs) is a significant step of osteogenic processes. The Wnt pathways has been identified as contributing to the regulation of osteogenic mineralization during development and disease. However, it remains unknown whether these MSCs in the vascular calcification differentiate into normal vascular smooth muscle cells (VSMCs) in vivo in order to treat damaged vascular tissue or into calcified VSMCs to aggravate calcification correlated to the Wnt pathways. Thus, it is necessary to analyze the mechanisms of MSC differentiation in detail. In the present study a cell-cell co-culturing in vitro system was used to observe MSCs that directly interact with normal or calcified VSMCs during calcification and to investigate the gene expression of the Wnt pathways during the process. Direct co-cultures were established by seeding two different cell types, VSMCs or calcified VSMCs, or a mixture of both at ratios of 5,000:5,000 cells/1.7 cm2 onto either gelatin-coated 1.7-cm2 chamber slides for immunohistochemical analysis or gelatin-coated 75-cm2 tissue culture flasks for protein or RNA isolation. Osteoblastic differentiation was evaluated by examining the cell morphology and assessing the activity of alkaline phosphatase in the cell lysates by alkaline phosphatase staining. Additionally, the mRNA expression levels of the genes encoding for proteins involved in the Wnt signaling proteins, Wnt5A, LRP6, Ror2, c-Jun-N-terminal kinase and β-catenin, were assessed in each group. The present study demonstrated that Wnts are expressed in the progress of differentiation of MSCs during calcification. MSCs can differentiate into different cell phenotypes when there is direct cell-cell contact with VSMCs or calcified VSMCs, and the Wnt5a/Ror2 signaling pathway may be associated with the determination of differentiation of MSCs in this process.