title: Ulinastatin inhibits oxidant-induced endothelial hyperpermeability and apoptotic signaling.
abstract: Oxidants are important signaling molecules known to increase endothelial permeability. Studies implicate reactive oxygen species (ROS) and the intrinsic apoptotic signaling cascades as mediators of vascular hyperpermeability. Here we report the protective effects of ulinastatin, a serine protease inhibitor with antiapoptotic properties, against oxidant-induced endothelial monolayer hyperpermeability. HUVECs were respectively pretreated with 10,000 and 50,000 u/l ulinastatin, followed by stimulation of 0.6 mM H₂O₂. Monolayer permeability was determined by transendothelial electrical resistance (TER); Mitochondrial release of cytochrome c was determined by enzyme-linked immunosorbent assay; Caspase-3 activity was measured by fluorometric assay; Adherens junction protein β-catenin was detected by immunofluorescense staining; Ratio of cell apoptosis was evaluated by Annexin-V/PI double stain assay; Mitochondrial membrane potential (Δψm) was determined with JC-1; Intracellular ATP content was assayed by a commercial kit; Bax and Bcl-2 expression were estimated by western blotting; Intracellular reactive oxygen species (ROS) level was measured by DCFH-DA. H₂O₂ exposure resulted in endothelial hyperpermeability and ROS formation (P < 0.05). The activation of mitochondrial intrinsic apoptotic signaling pathway was evidenced from BAX up-regulation, Bcl-2 down-regulation, mitochondrial depolarization, an increase in cytochrome c release, and activation of caspase-3 (P < 0.05). UTI (50,000 u/l) attenuated endothelial hyperpermeability, ROS formation, mitochondrial dysfunction, cytochrome c release, activation of caspase-3, and disruption of cell adherens junctions (P < 0.05). Together, these results demonstrate that UTI provides protection against vascular hyperpermeability by modulating the intrinsic apoptotic signaling.
authors.affiliation: Department of Critical Care Medicine, Nanfang Hospital, Southern Medical University Guangzhou, China ; Department of Critical Care Medicine, The First People's Hospital of Chenzhou, Institute of Translation Medicine Chenzhou, China.
authors.forename: Guicheng
authors.initials: G
authors.lastname: Li
authors.affiliation: Department of Critical Care Medicine, The First People's Hospital of Chenzhou, Institute of Translation Medicine Chenzhou, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Li
authors.affiliation: Department of Critical Care Medicine, The First People's Hospital of Chenzhou, Institute of Translation Medicine Chenzhou, China.
authors.forename: Yunfeng
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Critical Care Medicine, Nanfang Hospital, Southern Medical University Guangzhou, China.
authors.forename: Shumin
authors.initials: S
authors.lastname: Cai
authors.affiliation: Department of Anesthesiology, The First People's Hospital of Chenzhou, Institute of Translation Medicine Chenzhou, China.
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Department of Critical Care Medicine, Nanfang Hospital, Southern Medical University Guangzhou, China ; Guangdong Key Lab of Shock and Microcirculation Research, Department of Pathophysiology, Southern Medical University Guangzhou, China.
authors.forename: Zhenhua
authors.initials: Z
authors.lastname: Zeng
authors.affiliation: Department of Pathology, Maternal and Child Health Hospital of Liuzhou Liu Zhou, China.
authors.forename: Xingmin
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Critical Care Medicine, Nanfang Hospital, Southern Medical University Guangzhou, China ; Department of Anesthesiology, The First Affiliated Hospital of Fujian Medical University Fuzhou, China.
authors.forename: Youguang
authors.initials: Y
authors.lastname: Gao
authors.affiliation: Department of Critical Care Medicine, The First People's Hospital of Chenzhou, Institute of Translation Medicine Chenzhou, China.
authors.forename: Yunfeng
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Critical Care Medicine, Nanfang Hospital, Southern Medical University Guangzhou, China.
authors.forename: Zhongqing
authors.initials: Z
authors.lastname: Chen
mesh_terms: D022005:Adherens Junctions / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D001562:Benzimidazoles; D002232:Carbocyanines; D053148:Caspase 3 / Q000187:drug effects / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects*; D045304:Cytochromes c / Q000378:metabolism; D015536:Down-Regulation; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000494:pharmacology*; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*; D015854:Up-Regulation; D051176:beta Catenin / Q000378:metabolism
chemical_list: D001562:Benzimidazoles; D002232:Carbocyanines; D006023:Glycoproteins; D017382:Reactive Oxygen Species; D015842:Serine Proteinase Inhibitors; D051176:beta Catenin; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D045304:Cytochromes c; D053148:Caspase 3; C028665:urinastatin
keywords: Endothelial barrier; adherens junctions; apoptosis; mitochondria; oxidative stress