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

Abstract

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 MSC-induced 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.

Figure 1

Marrow stromal cell treatment of stroke rats decrease BBB leakage and increase Ang1, Tie2, and occludin expression in the ischemic brain: MCAo rats were treated with or without MSC after stroke. (A, D, G, and J) BBB leakage measured by Evans blue (A), and Ang1 (D), Tie2 (G), and occludin (J) expression in MCAo control rats, respectively. (B, E, H, and K) Evans blue (B), Ang1 (E), Tie2 (H), and occludin (K) expression in the MSC-treated rats, respectively. (C, F, I, and L) Quantitative data of Evans blue level in the ischemic brain (C), and Ang1 (F), Tie2 (I), and occludin (L)-positive cells percentage of area in the ischemic border. Marrow stromal cell treatment stroke animals significant decrease BBB leakage and increase the expression of Ang1, Tie2, and occludin compared with control MCAo animals. Scale bar (E, H, and K)=50μm. (M to R) Double immunostaining of Ang1 with astrocyte (M, Ang1; N, GFAP; and O, Ang1 with GFAP merged); Ang1 with pericyte (P, Ang1; Q, α-SMA; R, Ang1 with α-SMA merged). (S to X) Double immunostaining Tie2 with pericyte (S, Tie2; T, α-SMA; and U, Tie2 with á-SMA merged); Tie2 with brain endothelial cell (V, Tie2;W, vWF; and X, Tie2 with vWF merged), respectively.

Figure 2

Marrow stromal cells cocultured with MBECs or astrocytes regulates MBECs or astrocytes Ang1/Tie2 and VEGF/Flk1 gene and protein expression. (A and B) Gene expression measured by real-time PCR. (C)Protein expression measured by Western blot at 24 h. (D and E)Quantitative data of Western blot.

Figure 3

Marrow stromal cells promotes capillary tube formation in vitro. (A) Quantitative data of capillary tube formation at 5 h after culture. (B to F) Tube formation in conditioned media from control (C), MSCs culture alone (D), MSCs cocultured with MBECs (E), and MSCs cocultured with astrocytes (F). (B) quantitative data of capillary tube formation at 24 h after culture. Scale bar in (F)=100 μm.

Figure 4

Knockdown Tie2 expression in MBECs decreases Tie2 gene and protein expression and attenuates MSC-induced capillary tube formation. (A) Tie2 mRNA expression in MBECs measured by real-time PCR. (B) Tie2 protein expression in MBECs measured by Western blot. (C to F)Capillary tube-like formation in normal MBECs (C), normal MBECs treated with MSC-conditioned media (D), Tie2 knockdown MBECs control (E), and Tie2 knockdown MBECs treated with conditioned MSC media (F). (G)Quantitative analysis of capillary-like tube formation. Scale bar in (F)=100 μm.