Two dimensional (2D) co-cultures of human bone marrow stromal cells (HBMSCs) and human umbilical vein endothelial cells (HUVECs) stimulate osteoblastic differentiation of HBMSCs, induce the formation of self-assembled network and cell interactions between the two cell types involving many vascular molecules. Because of their strong activities on angiogenesis and tissue remodeling, urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), matrix metalloproteinase-2 (MMP-2) as well tissue inhibitors of matrix metalloproteinase-2 (TIMP-2) were investigated in this 2D co-culture model. We found that the expression of uPA, MMP-2 in the co-cultured cells was significantly higher than those in mono-cultured cells. In opposite, PAI-1, expressed only by HUVECs is not regulated in the co-culture. Inhibition assays confirm that uPA played a critical role in the formation of self-assembled network as neutralization of uPA disturbed this network. In the same context, inhibition of MMP-2 prevented the formation of self-assembled network, while the inhibition of uPA abolished the over expression and the activity of MMP-2. This upregulation could initiate the uPA expression and proteolysis processes through the MMP-2 activity, and may contribute to endothelial cell migration and the formation of this self-assembled network observed in these 2D co-cultured cells.
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