Induction of Tie-1 and Tie-2 receptor protein expression after cerebral ischemia-reperfusion

Tie-1 and Tie-2 are receptor tyrosine kinases (RTKs) that are exclusively e xpressed in endothelial cells and play important roles in endothelial cell biology. The authors have reported previously the temporal profiles of Tie- 1 and Tie-2 mRNA expression after focal cerebral ischemia-reperfusion. in t he current study, the localization of Tie-1/Tie-2 mRNA and proteins were fu rther investigated in the same focal ischemia model. In situ hybridization showed that, after 60-minute ischemia and 72-hour reperfusion, both Tie-1 a nd Tie-2 mRNA appeared as capillary-like structures in the ischemic middle cerebral artery (MCA) cortex. Western blot analysis showed a biphasic expre ssion of Tie-1 protein in the same region. The first peak, spanning the isc hemic and early reperfusion period, was of low intensity and short-lived. T he second peak was of greater intensity and spanning the period from 72 to 168 hours after reperfusion. Similarly, Tie-2 expression at the protein lev el also exhibited a biphasic pattern. Immunohistochemical studies. after 72 hours of reperfusion, showed that although Tie-1 and Tie-2 were detected w ithin the ischemic cortex, they actually were expressed in different popula tions of endothelial cells in different regions. In agreement with the insi tu hybridization study, Tie-1 immunoreactivity appeared as capillary-like s tructures in cortical layers 2 to 4. Similar capillary-like appearance of T ie-2 immunoreactivity was noted in the outer cortical layers. In addition, Tie-2 immunoreactivity also was observed in cortical layer 6b, where de nov o large vessel formation was noted. Cellular colocalization experiments rev ealed that Tie-2 is expressed in proximity to its antagonist, Angpo-2, as w ell as basic fibroblast growth factor (bFGF) and vascular endothelial growt h factor (VEGF) in cortical layer 1, where active vessel remodeling was not ed. Interestingly, bFGF only partially colocalized with VEGF, suggesting di fferential roles for these angiogenic factors during vessel remodeling. Tie -1 protein, to a lesser degree, also colocalized with Angpo-2, bFGF, and VE GF in cortical layer 1. Magnetic resonance imaging (MRI) showed increased r egional cerebral blood flow (CBF) corresponding to the expression of these angiogenesis gene products. Together, these findings suggest that the evolv ing expression of angiogenesis genes underlie the robust vascular remodelin g after ischemia and reperfusion.