Although the source of the VEGF increased by NPCs remains to be determined,

it has been found that treatment of astrocytes with bone marrow-derived stromal cells stimulates the expression of VEGF in the astrocytes (Zacharek et al. 2007). In this regard, it is interesting to note that GFP-positive cells, which were the NPCs injected, had differentiated into GFAP-positive cells in the peri-infarcted areas by day 28. In this study, the level of Ang-1 after the embolism was time-dependently increased. In addition, the expression Inhibitors,research,lifescience,medical of Ang-1 was enhanced by injection of NPCs, whereas the level of Ang-2 was unchanged by NPCs compared with that of vehicle-injected ME rats on day 28. Ang-1 function is an agonist for Tie2, whereas Ang-2

is antagonistic toward Tie2. Therefore, the shift of the balance between Ang-1 and Ang-2 has been implicated in enhanced angiogenic activity after an embolism. As Tie2 is expressed predominantly in endothelial cells, we further examined the level of Tie2 protein Inhibitors,research,lifescience,medical in the brain capillaries isolated from microsphere-embolized rats. The NPC-induced increase in Tie2 proteins in brain capillaries on day 28 may have Inhibitors,research,lifescience,medical contributed to the ability of NPCs to enhance blood vessel formation and maintenance for a long period after the embolism. Ang-1 induces the gene expression of occludin, which is one of the tight junctional proteins in brain capillary endothelial cells, by acting through Tie2 (Lee et al. 2003; Hori et al. 2004). It is noteworthy that the injection of recombinant human Ang-1 increases the expression of occludin and ZO-1 in the ischemic brain (Yu et al. 2012). Therefore, Inhibitors,research,lifescience,medical the relatively high level of Ang-1 on day 28 after the NPC injection compared with that of Ang-2 might have promoted the expression Inhibitors,research,lifescience,medical of occludin and ZO-1 in brain capillary endothelial cells, which proteins have been implicated in vessel formation and vascular stabilization.

This interpretation is consistent with the finding that the injection of NPCs tended to increase the amount of ZO-1 protein in brain capillaries on day 28 after the embolism. In conclusion, as marked expression of Ang-1 after injection heptaminol of NPCs occurred in the peri-infarct area on day 28, intravenous injection of NPCs had the ability to promote angiogenesis for a long period through Ang-1/Tie2 and/or VEGF/VEGFR2 signaling even when the injection was started on day 7 after the embolism. These NPC-induced angiogenic activities may be involved in vessel stabilization and maintenance as well as in vessel formation for a long period after the embolism. They may be associated with the improvement of brain dysfunction after cerebral embolism, including learning and memory dysfunction and depression-like behavior. Acknowledgments This research was supported in part by Takeda Science Foundation. Conflict of Forskolin mouse Interest None declared.