Childs Ed W, Tharakan Binu, Byrge Nickolas, Tinsley John H, Hunter Felicia A, Smythe W Roy
Department of Surgery, Texas A&M University System HSC College of Medicine, Scott & White Memorial Hospital, 2401 South 31st Street, Temple, TX 76508, USA.
Am J Physiol Heart Circ Physiol. 2008 May;294(5):H2285-95. doi: 10.1152/ajpheart.01361.2007. Epub 2008 Mar 14.
Studies from our laboratory demonstrated the involvement of intrinsic apoptotic signaling in hyperpermeability following hemorrhagic shock (HS). Angiopoietin 1 (Ang-1), a potent inhibitor of hyperpermeability, was recently shown to inhibit apoptosis. The purpose of our study was to determine the effectiveness of Ang-1 in attenuating HS-induced hyperpermeability and its relationship to apoptotic signaling. HS was induced in rats by withdrawing blood to reduce the mean arterial pressure to 40 mmHg for 1 h, followed by reperfusion. Mesenteric postcapillary venules were examined for changes in hyperpermeability by intravital microscopy. Mitochondrial release of second mitochondrial derived activator of caspases (smac) and cytochrome c were determined by Western blot and ELISA, respectively. Caspase-3 activity was determined by fluorometric assay. Parallel studies were performed in rat lung microvascular endothelial cell (RLMEC) monolayers, utilizing HS serum and the proapoptotic Bcl-2 homologous antagonist/killer [BAK (BH3)] peptide as inducers of hyperpermeability. In rats, Ang-1 (200 ng/ml) attenuated HS-induced hyperpermeability versus the HS group (P < 0.05). Ang-1 prevented HS-induced collapse of mitochondrial transmembrane potential (DeltaPsi(m)), smac and cytochrome c release, and caspase-3 activity (P < 0.05). In RLMEC monolayers, HS serum and BAK (BH3) peptide both induced hyperpermeability that was inhibited by Ang-1 (P < 0.05). Ang-1 attenuated HS and BAK (BH3) peptide-induced collapse of DeltaPsi(m), smac release, cytochrome c release, activation of caspase-3, and vascular hyperpermeability. In vivo, BAK (BH3) induced vascular hyperpermeability that was attenuated by Ang-1 (P < 0.05). These findings suggest that Ang-1's role in maintaining microvascular endothelial barrier integrity involves the intrinsic apoptotic signaling cascade.
我们实验室的研究表明，内源性凋亡信号参与了失血性休克（HS）后的高通透性过程。血管生成素1（Ang-1）是一种有效的高通透性抑制剂，最近研究表明它可抑制细胞凋亡。本研究的目的是确定Ang-1在减轻HS诱导的高通透性方面的有效性及其与凋亡信号的关系。通过放血使大鼠平均动脉压降至40 mmHg持续1小时，随后进行再灌注，从而诱导大鼠发生HS。通过活体显微镜检查肠系膜毛细血管后微静脉的高通透性变化。分别通过蛋白质免疫印迹法和酶联免疫吸附测定法测定半胱天冬酶-3（caspase-3）激活剂（smac）和细胞色素c从线粒体的释放情况。通过荧光测定法测定caspase-3活性。利用HS血清和促凋亡的Bcl-2同源拮抗剂/杀手[BAK（BH3）]肽作为高通透性诱导剂，在大鼠肺微血管内皮细胞（RLMEC）单层中进行平行研究。在大鼠中，与HS组相比，Ang-1（200 ng/ml）减轻了HS诱导的高通透性（P < 0.05）。Ang-1可防止HS诱导的线粒体跨膜电位（ΔΨm）崩溃、smac和细胞色素c释放以及caspase-3活性（P < 0.05）。在RLMEC单层中，HS血清和BAK（BH3）肽均诱导了高通透性，而Ang-1可抑制这种高通透性（P < 0.05）。Ang-1减轻了HS和BAK（BH3）肽诱导的ΔΨm崩溃、smac释放、细胞色素c释放、caspase-3激活以及血管高通透性。在体内，BAK（BH3）诱导的血管高通透性被Ang-1减轻（P < 0.05）。这些发现表明，Ang-1在维持微血管内皮屏障完整性中的作用涉及内源性凋亡信号级联反应。
