Angiopoietin1/Tie2 and VEGF/Flk1 induced by MSC treatment amplifies angiogenesis and vascular stabilization after stroke

Bone marrow stromal cells (MSCs) increase vascular endothelial growth factor ( VEGF) expression and promote angiogenesis after stroke. Angiopoietin-1 (Ang1) and its receptor Tie2 mediate vascular integrity and angiogenesis as does VEGF and its receptors. In this study, we tested whether MSC treatment of stroke increases Ang1/Tie2 expression, and whether Ang1/Tie2 with VEGF/vascular endothelial growth factor receptor 2 (VEGFR2) (Flk1), in combination, induced by MSCs enhances angiogenesis and vascular integrity. Male Wistar rats were subjected to middle cerebral artery occlusion (MCAo) and treated with or without MSCs. Marrow stromal cell treatment significantly decreased blood-brain barrier (BBB) leakage and increased Ang1, Tie2, and occludin ( a tight junction protein) expression in the ischemic border compared with MCAo control. To further test the mechanisms of MSC-induced angiogenesis and vascular stabilization, cocultures of MSCs with mouse brain endothelial cells (MBECs) or astrocytes were performed. Supernatant derived from MSCs cocultured with MBECs significantly increased MBEC expression of Ang1/ Tie2 and Flk1 compared with MBEC alone. Marrow stromal cells cocultured with astrocytes also significantly increased astrocyte VEGF and Ang1/ Tie2 expression compared with astrocyte culture alone. Conditioned media from MSCs alone, and media from cocultures of MSCs with astrocytes or MBECs, all significantly increased capillary tube- like formation of MBEC compared with control Dulbecco's modified Eagle's medium media. Inhibition of Flk1 and/ or Ang1 significantly decreased MSCinduced MBEC tube formation. Knockdown of Tie2 expression in MBECs significantly inhibited MSC- induced tube formation. Our data indicate MSC treatment of stroke promotes angiogenesis and vascular stabilization, which is at least partially mediated by VEGF/ Flk1 and Ang1/ Tie2.