Division of Stroke, University of Nottingham, Nottingham, UK.
Stroke. 2010 Sep;41(9):2056-63. doi: 10.1161/STROKEAHA.109.574939. Epub 2010 Jul 22.
Enhanced vascular permeability attributable to disruption of blood-brain barrier results in the development of cerebral edema after stroke. Using an in vitro model of the brain barrier composed of human brain microvascular endothelial cells and human astrocytes, this study explored whether small GTPase RhoA and its effector protein Rho kinase were involved in permeability changes mediated by oxygen-glucose deprivation (OGD), key pathological phenomena during ischemic stroke.
OGD increased RhoA and Rho kinase protein expressions in human brain microvascular endothelial cells and human astrocytes while increasing or unaffecting that of endothelial nitric oxide synthase in respective cells. Reperfusion attenuated the expression and activity of RhoA and Rho kinase in both cell types compared to their counterparts exposed to equal periods of OGD alone while selectively increasing human brain microvascular endothelial cells endothelial nitric oxide synthase protein levels. OGD compromised the barrier integrity as confirmed by decreases in transendothelial electric resistance and concomitant increases in flux of permeability markers sodium fluorescein and Evan's blue albumin across cocultures. Transfection of cells with constitutively active RhoA also increased flux and reduced transendothelial electric resistance, whereas inactivation of RhoA by anti-RhoA Ig electroporation exerted opposite effects. In vitro cerebral barrier dysfunction was accompanied by myosin light chain overphosphorylation and stress fiber formation. Reperfusion and treatments with a Rho kinase inhibitor Y-27632 significantly attenuated barrier breakdown without profoundly altering actin structure.
Increased RhoA/Rho kinase/myosin light chain pathway activity coupled with changes in actin cytoskeleton account for OGD-induced endothelial barrier breakdown.
血脑屏障的破坏导致血管通透性增强，进而引发中风后的脑水肿。本研究采用由人脑微血管内皮细胞和星形胶质细胞组成的体外血脑屏障模型，探讨小 GTP 酶 RhoA 及其效应蛋白 Rho 激酶是否参与了脑缺血中风过程中的关键病理现象——氧葡萄糖剥夺（OGD）所介导的通透性变化。
OGD 增加了人脑微血管内皮细胞和星形胶质细胞中 RhoA 和 Rho 激酶蛋白的表达，同时增加或不影响各自细胞中内皮型一氧化氮合酶的表达。与单独接受同等 OGD 时间的对照相比，再灌注可降低两种细胞类型中 RhoA 和 Rho 激酶的表达和活性，同时选择性增加人脑微血管内皮细胞内皮型一氧化氮合酶蛋白水平。OGD 破坏了屏障的完整性，这可通过跨内皮电阻的降低和通透性标志物荧光素钠和伊文思蓝白蛋白的通量增加来证实。转染细胞使其表达组成型激活的 RhoA 也会增加通量并降低跨内皮电阻，而用抗 RhoA Ig 电穿孔使 RhoA 失活则会产生相反的效果。体外脑屏障功能障碍伴随着肌球蛋白轻链过度磷酸化和应力纤维形成。再灌注和 Rho 激酶抑制剂 Y-27632 的处理显著减轻了屏障破坏，而对肌动蛋白结构没有显著改变。
RhoA/Rho 激酶/肌球蛋白轻链通路活性的增加以及肌动蛋白细胞骨架的变化是 OGD 诱导的内皮屏障破坏的原因。
