Effects of Angiopoietin-1 on Hemorrhagic Transformation and Cerebral Edema after Tissue Plasminogen Activator Treatment for Ischemic Stroke in Rats

An angiogenesis factor, angiopoietin-1 (Ang1), is associated with the blood-brain barrier (BBB) disruption after focal cerebral ischemia. However, whether hemorrhagic transformation and cerebral edema after tissue plasminogen activator (tPA) treatment are related to the decrease in Ang1 expression in the BBB remains unknown. We hypothesized that administering Ang1 might attenuate hemorrhagic transformation and cerebral edema after tPA treatment by stabilizing blood vessels and inhibiting hyperpermeability. Sprague-Dawley rats subjected to thromboembolic focal cerebral ischemia were assigned to a permanent ischemia group (permanent middle cerebral artery occlusion; PMCAO) and groups treated with tPA at 1 h or 4 h after ischemia. Endogenous Ang1 expression was observed in pericytes, astrocytes, and neuronal cells. Western blot analyses revealed that Ang1 expression levels on the ischemic side of the cerebral cortex were decreased in the tPA-1h, tPA-4h, and PMCAO groups as compared to those in the control group (P = 0.014, 0.003, and 0.014, respectively). Ang1-positive vessel densities in the tPA-4h and PMCAO groups were less than that in the control group (p = 0.002 and <0.001, respectively) as well as that in the tPA-1h group (p = 0.047 and 0.005, respectively). These results suggest that Ang1-positive vessel density was maintained when tPA was administered within the therapeutic time window (1 h), while it was decreased when tPA treatment was given after the therapeutic time window (4 h). Administering Ang1 fused with cartilage oligomeric protein (COMP) to supplement this decrease has the potential to suppress hemorrhagic transformation as measured by hemoglobin content in a whole cerebral homogenate (p = 0.007) and cerebral edema due to BBB damage (p = 0.038), as compared to administering COMP protein alone. In conclusion, Ang1 might be a promising target molecule for developing vasoprotective therapies for controlling hemorrhagic transformation and cerebral edema after tPA treatment.

血管生成素-1（angiopoietin-1, Ang1）作为一类血管生成因子，与局灶性脑缺血后的血脑屏障（blood-brain barrier, BBB）破坏存在关联。然而，接受组织型纤溶酶原激活剂（tissue plasminogen activator, tPA）治疗后出现的出血性转化与脑水肿，是否与血脑屏障内Ang1表达下调相关，目前仍未明确。本研究提出假说：通过稳定血管、抑制血管高通透性，外源性给予Ang1可减轻tPA治疗后的出血性转化与脑水肿。
本研究将接受血栓栓塞性局灶性脑缺血造模的斯普拉格-道利（Sprague-Dawley, SD）大鼠分为三组：永久性缺血组（永久性大脑中动脉闭塞，permanent middle cerebral artery occlusion, PMCAO），以及缺血后1小时或4小时接受tPA治疗的两个干预组。研究观察到，周细胞、星形胶质细胞与神经元细胞中均存在内源性Ang1的表达。蛋白质免疫印迹（Western blot）分析结果显示，与对照组相比，tPA-1h组、tPA-4h组与PMCAO组大鼠大脑皮层缺血侧的Ang1表达水平均显著下调（P值分别为0.014、0.003与0.014）。tPA-4h组与PMCAO组的Ang1阳性血管密度均低于对照组（P值分别为0.002与<0.001），同时也低于tPA-1h组（P值分别为0.047与0.005）。上述结果表明，若在治疗时间窗（1小时）内给予tPA，Ang1阳性血管密度可维持稳定；但若在治疗时间窗外（4小时）给予tPA，则Ang1阳性血管密度会出现下降。
与单独给予软骨寡聚基质蛋白（cartilage oligomeric protein, COMP）相比，给予融合了COMP的Ang1以补充该表达下调，可通过检测全脑匀浆的血红蛋白含量（P=0.007）与血脑屏障损伤引发的脑水肿程度（P=0.038），有效抑制出血性转化。
综上，Ang1有望成为开发血管保护性治疗方案的潜在靶标分子，用于调控tPA治疗后的出血性转化与脑水肿。