Alluri Himakarnika, Stagg Hayden W, Wilson Rickesha L, Clayton Robert P, Sawant Devendra A, Koneru Madhavi, Beeram Madhava R, Davis Matthew L, Tharakan Binu
Departments of Surgery and Pediatrics, Texas A&M University Health Science Center College of Medicine and Scott & White Healthcare, Temple, Texas, USA.
Microcirculation. 2014 Feb;21(2):187-95. doi: 10.1111/micc.12110.
Microvascular hyperpermeability that occurs due to breakdown of the BBB is a major contributor of brain vasogenic edema, following IR injury. In microvascular endothelial cells, increased ROS formation leads to caspase-3 activation following IR injury. The specific mechanisms, by which ROS mediates microvascular hyperpermeability following IR, are not clearly known. We utilized an OGD-R in vitro model of IR injury to study this.
RBMEC were subjected to OGD-R in presence of a caspase-3 inhibitor Z-DEVD, caspase-3 siRNA or an ROS inhibitor L-AA. Cytochrome c levels were measured by ELISA and caspase-3 activity was measured fluorometrically. TJ integrity and cytoskeletal assembly were studied using ZO-1 immunofluorescence and rhodamine phalloidin staining for f-actin, respectively.
OGD-R significantly increased monolayer permeability, ROS formation, cytochrome c levels, and caspase-3 activity (p < 0.05) and induced TJ disruption and actin stress fiber formation. Z-DEVD, L-AA and caspase-3 siRNA significantly attenuated OGD-R-induced hyperpermeability (p < 0.05) while only L-AA decreased cytochrome c levels. Z-DEVD and L-AA protected TJ integrity and actin cytoskeletal assembly.
These results suggest that OGD-R-induced hyperpermeability is ROS and caspase-3 dependent and can be regulated by their inhibitors.
血脑屏障（BBB）破坏所致的微血管通透性增加是缺血再灌注（IR）损伤后脑血管源性水肿的主要原因。在微血管内皮细胞中，IR损伤后活性氧（ROS）生成增加会导致半胱天冬酶-3（caspase-3）激活。目前尚不清楚ROS介导IR后微血管通透性增加的具体机制。我们利用IR损伤的氧糖剥夺复氧（OGD-R）体外模型对此进行研究。
将大鼠脑微血管内皮细胞（RBMEC）在存在半胱天冬酶-3抑制剂Z-DEVD、半胱天冬酶-3小干扰RNA（siRNA）或ROS抑制剂L-抗坏血酸（L-AA）的情况下进行OGD-R处理。通过酶联免疫吸附测定（ELISA）法检测细胞色素c水平，并用荧光法测定半胱天冬酶-3活性。分别使用紧密连接蛋白1（ZO-1）免疫荧光法和罗丹明鬼笔环肽染色法检测f-肌动蛋白（f-actin）来研究紧密连接（TJ）完整性和细胞骨架组装。
OGD-R显著增加单层通透性、ROS生成、细胞色素c水平和半胱天冬酶-3活性（p<0.05），并诱导TJ破坏和肌动蛋白应激纤维形成。Z-DEVD、L-AA和半胱天冬酶-3 siRNA显著减轻OGD-R诱导的通透性增加（p<0.05），而只有L-AA降低了细胞色素c水平。Z-DEVD和L-AA保护TJ完整性和肌动蛋白细胞骨架组装。
这些结果表明，OGD-R诱导的通透性增加是ROS和半胱天冬酶-3依赖性的，并且可以被其抑制剂调节。
