Abstract
Background: Annexin II (ANX2) is a fibrinolytic receptor that serves as a binding site for plasminogen and tissue plasminogen activator, facilitating the generation of plasmin. ANX2 is present on a wide variety of cells including vascular endothelial cells as well as macrophages. ANX2 has been shown to play a key role in extracellular matrix degradation, cellular migration, and invasion. This degradation of extracellular matrix may also cause the release of matrix-bound angiogenic factors such as VEGF and FGF. We hypothesized that adult human mesenchymal stem cells (hMSCs) express ANX2 and utilize this receptor for plasmin generation to facilitate basement membrane invasion.
Methods: Primary hMSCs were isolated from the sternal bone marrow of patients undergoing median sternotomy. Stem cell surface markers were characterized via immuno-fluorescence. The presence of ANX2 protein by hMSCs was established via western blot. ANX2 mediated plasminogen activation and plasmin generation was quantified using chromozyme-P as a colorimetric substrate. Invasion assays were performed in dual-chamber culture wells containing matrigel inserts. hMSCs were plated into upper chambers containing: serum-free medium (SFM), SFM + Plasminogen, or SFM + Plasminogen + epsilon-aminocaproic acid (e-ACA inhibits binding of plasminogen to ANX2). After 24 hours, invasive cells were isolated and counted.
Results: Sternal bone marrow derived hMSCs expressed the membrane phenotype CD34 (−), CD14 (−), CD44 (+), CD105 (+), CD106 (+). The presence of ANX2 was confirmed by western blot analysis. hMSCs generated 1.95 units of plasmin per milligram of protein. There was a 20% (p 0 .004) increase in hMSC invasion in the wells containing plasminogen as compared to SFM alone. When e-ACA was introduced there was a decrease in hMSC invasion back to control values.
Conclusion: Our observations establish for the first time the presence and functional activity of ANX2 in hMSCs. These data suggest that mesenchymal stem cell expression of ANX2 facilitates plasminogen-mediated hMSC trans-endothelial invasion, migration and the release of pro-angiogenic factors from within the extracellular matrix, promoting stem cell directed repair and angiogenesis.
Heterogeneous Differentiation of Human Mesenchymal Stem Cells in 3D Extracellular Matrix Composites
