Angiopoietin/Tie2 pathway mediates type 2 diabetes induced vascular damage after cerebral stroke

Abstract

We investigated the changes and the molecular mechanisms of cerebral vascular damage after stroke in type-2 diabetic (T2DM) mice. Adult male db/db T2DM and wild-type (WT) mice were subjected to transient middle cerebral artery occlusion (MCAo) and sacrificed 24 hours after MCAo. T2DM-mice exhibited significantly increased blood glucose, brain hemorrhagic rate, mortality and cerebrovascular density, but decreased cerebrovascular diameter, arteriolar density and arterial mural cell numbers in the ischemic brain compared with WT mice. The hemorrhagic rate was significantly correlated with the mortality (r = 0.85). T2DM-mice also exhibited increased blood-brain barrier leakage and concomitantly, increased Angiopoietin2, but decreased Angiopoietin1, Tie2 and tight junction protein expression in the ischemic brain. Angiopoietin1 gene expression also significantly decreased in the common carotid artery (CCA) in T2DM-mice compared with WT mice after stroke. To further test the effects of T2DM on cerebrovascular damage, we performed in vitro studies. The capillary-like tube formation of primary cultured mouse brain endothelial cells (MBECs) significantly increased, but artery cell migration in the primary CCA cultures significantly decreased both in Sham and MCAo T2DM-mice compared with the WT mice. Angiopoietin1 treatment significantly increased artery cell migration in T2DM-CCA after MCAo. Tie2-FC, a neutralized Tie2 antibody, significantly decreased artery cell migration in WT-CCA after MCAo. Therefore, decreased Angiopoietin1/Tie2 and increased Angiopoietin2 expression may contribute to diabetes-induced vascular damage after stroke.

Fig.1

T2DM increase BBB leakage and vascular density, decrease vascular/arteriolar diameter, arteriolar wall mural cell number, and decrease tight junction protein expression in the ischemic brain after stroke. A-C: BBB leakage and quantitative data measured by Evens Blue dye perfusion and extravasation, n=4/group; D-I: vWF immunostaining and quantitative data; J-O: αSMA immunostaining and quantitative data; P-T: Occludin immunofluorescent staining and quantitative data. n = 11/group, Scale bar in D, J and P = 40 μm.

Fig.2

T2DM decrease Ang1 and Tie2, increase Ang2 expression in the ischemic brain after stroke measured by immunostaining. A-E: Ang1 immunostaining and quantitative data; F-J: Ang2 immunostaining and quantitative data; K-O: Tie2 immunofluorescent staining and quantitative data. Scale bar in A = 40 μm, in F = 20 μm, in K = 50 μm. n = 11/group.

Fig.3

T2DM decrease Ang1 and Tie2, but increase Ang2 expression in the ischemic brain after stroke measured by Western blot, protein array and RT-PCR. A and B: Ang1, Ang2 and Tie2 protein expression in the ischemic ipsilateral and contralateral brain (A) and quantitative data (B) measured by Western blot assay; C: Ang1 expression in the ischemic ipsilateral and contralateral brain measured by protein array; D: Ang1, Ang2 and Tie2 gene expression in the ischemic ipsilateral brain measured by RT-PCR. n = 4/group.

Fig.4

T2DM decrease artery cell migration in the primary cultured artery both in Sham and MCAo group; Tie2-FC attenuate artery cell migration in WT-CCA; Ang1 treatment increase T2DM-artery cell migration after stroke. A-C: primary artery cell migration in CCA derived from Sham WT (A) and DM mice (B), and quantitative data (C); D-H: primary artery cell migration in CCA derived from WTMCAo (D), DM-MCAo (E), WT-MCAo + Tie2-FC 2 μg/ml (F), DM-MCAo + Ang1 100 ng/ml (G), and quantitative data (H); I: Ang1, Ang2 and Tie2 gene expression in the CCA derived from WT-MCAo and DM-MCAo measured by RT-PCR. n = 6/group.

Fig.5

T2DM increase capillary-like tube formation in the primary cultured MBEC both in Sham and MCAo group. A: WT-Sham, B: DM-Sham, C: WT-MCAo, D: DM-MCAo, E: quantitative data of tube formation. n = 6/group.