A mechanism for TNF induced endothelial dysfunction

TNF诱导内皮功能障碍的机制

• 批准号: 7161387
• 负责人: ARNOLD JOHNSON
• 金额: $ 11.64万
• 依托单位: ALBANY RESEARCH INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7161387
• 关键词: 3-nitrotyrosine; 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one; Actin-Binding Protein; Actins; Address; Adenovirus Vector; Adenoviruses; Adherence; Adherens Junction; Adult; Adult Respiratory Distress Syndrome; Apoptosis; Appendix; Back; Binding; Biological Assay; Carbohydrates; Cell Fraction; Cell Line; Cells; Complex; Critiques; Cytoskeleton; Data; Detergents; Endothelial Cells; Event; Excision; F-Actin; Facility Construction Funding Category; Functional disorder; G Actin; Gene Transfer; Generations; Genes; Immunochemistry; Inflammation; Injury; Intercellular Junctions; Interphase Cell; Knock-out; Knockout Mice; Light; Lipids; Literature; Lung; Maintenance; Manuscripts; Mediating; Mediator of activation protein; Membrane; Mitogen-Activated Protein Kinases; Morphology; Mus; Mutation; Myosin Light Chain Kinase; NADPH Oxidase; Newborn Respiratory Distress Syndrome; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Pathway interactions; Peripheral; Permeability; Peroxonitrite; Phosphorylation; Phosphotransferases; Physiological; Polymers; Protein Kinase C; Protein Tyrosine Kinase; Proteins; Protocols documentation; Reaction; Recovery; Research Design; Role; Second Messenger Systems; Sedimentation process; Sepsis Syndrome; Signal Pathway; Signal Transduction; Signaling Molecule; Superoxides; Syndrome; TNF gene; Technology; Testing; Tight Junctions; Time; Transfection; Tyrosine; alpha catenin; beta Actin; expression vector; human AKAP13 protein; improved; in vivo; inhibitor/antagonist; monolayer; monomer; nitrate; nitration; novel; numb protein; occludin; polymerization; prevent; protein protein interaction; pulmonary vascular permeability; research study; response; second messenger; size; transgene expression; vector

DESCRIPTION (provided by applicant): Statement of the Problem: TNF is a primary mediator of sepsis syndrome, systemic inflammation syndrome (SIS) and the adult respiratory distress syndrome (ARDS). TNF induces an increase in pulmonary vascular permeability in vivo, in the isolated lung and in pulmonary endothelial monolayers. There are many signal-pathways that are modulated in response to TNF such as different isoforms of protein kinase C (PKCs), myosin light chain kinase (MLCK), Mitogen Activated Protein Kinases (MAPKs), tyrosine kinases and capases of apoptosis. However, the role of these pathways in endothelial injury in response to TNF is still not very clear. Our studies indicate that PKCalpha, endothelial nitric oxide synthetase (eNOS) and NADPH-oxidase mediate the response to TNF; however, the paradigm that integrates PKCalpha, eNOS and NADPH-oxidase is not known. Our studies will show that a convergence pathway for PKCalpha , eNOS and NADPH-oxidase is peroxynitrite (ONOO_), formed by the interaction of nitric oxide (.NO) with superoxide (.O2), Our proposal will show that the endothelial barrier dysfunction in response to TNF is induced, at least in part, by the ONOO_ mediated nitration of critical tyrosine residues in cortical actin (i.e., nitrotyrosine-beta-actin). The nitrotyrosine-beta-actin polymers do not complex with actin-binding proteins of the zonular adherence (e.g., alpha-catenin) and/or zonular occludin (e.g., ZO-1) proteins, effectively enough, to maintain barrier function. The nitrotyrosine-beta-actin formation is because of an increase in ONOO( .NO+.O2 ->ONOO_) generated in the peripheral membrane. A novel mechanism for the increases in endothelial permeability in response to TNF will be investigated in this proposal. Hypothesis: The hypothesis of this proposal is that nitration of critical tyrosine residues in cortical-actin (i.e., nitrotyrosine-beta-actin) mediates, at least in part, the endothelial barrier dysfunction in response to TNF. Nitrotyrosine-beta-actin will not promote maintenance of F-actin polymers because of "capping-nitration" of the F-actin barbed ends, and the slow incorporation of the nitrotyrosine-beta-actin G-monomers into F-actin. Subsequently, the nitrotyrosine-beta-actin polymers do not complex with the actinbinding proteins of the zonular adherence and/or zonular occludin effectively enough to maintain barrier function. The nitrotyrosine-beta-actin formation is because of an increase in ONOO_ generation near the cortical beta-actin. The ONOO_ is generated from the reaction of .NO with .O2_ because of activation of NADPH-oxidase and eNOS, respectively, in the peripheral membrane. The activation of NADPH-oxidase is because of PKC" dependent phosphorylation of p47 phox. A novel mechanism for the increases in endothelial permeability in response to TNF will be investigated in this proposal. The Specific Objectives are: (1) To determine if PKCalpha-induced NADPH-oxidase activation mediates the ONOO_-induced change in nitrated-actin and actin morphology in pulmonary microvessel endothelial cell monolayers (PMEM) in response to TNF (Yearsl-2). (2) To determine which tyrosine residues of actin are nitrated that permit the change in actin morphology and increase in endothelial permeability in PMEM in response to TNF (Years 2-3) (3). To determine if nitrotyrosine-beta-actin mediates an increase in endothelial permeability because of a change in the incorporation of beta-actin with the actin-binding proteins of the zonular adherence and zonular occludin junctions, (-catenin and ZO-1, respectively (Years 3-4).

描述（由申请人提供）：问题的说明：TNF是败血症综合征，全身性炎症综合征（SIS）和成人呼吸窘迫综合征（ARDS）的主要介体。 TNF在分离的肺和肺部内皮单层中诱导体内肺血管通透性的增加。有许多对TNF的调节，例如蛋白激酶C（PKCS），肌球蛋白轻链激酶（MLCK），有丝分裂原活化蛋白激酶（MAPK），酪氨酸激酶，酪氨酸激酶和凋亡的囊酶。但是，这些途径在响应TNF的内皮损伤中的作用仍然不太清楚。我们的研究表明，PKCALPHA，内皮一氧化氮合成酶（ENOS）和NADPH-氧化酶介导对TNF的反应。但是，尚不清楚整合PKCALPHA，ENOS和NADPH-氧化酶的范例。 Our studies will show that a convergence pathway for PKCalpha , eNOS and NADPH-oxidase is peroxynitrite (ONOO_), formed by the interaction of nitric oxide (.NO) with superoxide (.O2), Our proposal will show that the endothelial barrier dysfunction in response to TNF is induced, at least in part, by the ONOO_ mediated nitration of critical皮质肌动蛋白（即硝基酪氨酸β-肌动蛋白）中的酪氨酸残基。硝基酪氨酸-β-肌动蛋白聚合物与肌动蛋白结合蛋白（例如α-蛋白蛋白）和/或扎合occludin（例如ZO-1）蛋白有效地足以维持屏障的功能，与肌动蛋白结合蛋白（例如α-蛋白酶）和/或扎原闭合蛋白不复杂。硝基酪氨酸β-肌动蛋白的形成是由于外周膜中产生的Onoo（.no+.o2-> onoo_）的增加。该提案将研究一种响应TNF的内皮渗透性增加的新型机制。假设：该提议的假设是，皮质 - 肌动蛋白（即硝基酪氨酸-β-肌动蛋白）中临界酪氨酸残基的硝化作用至少部分是内皮屏障功能障碍，响应于TNF。由于F-肌动蛋白带刺的末端“限制核化”，硝基酪氨酸β-肌动蛋白不会促进F-肌动蛋白聚合物的维持，而硝基酪氨酸-β-肌动蛋白G-M-Manoners慢慢掺入F-肌动蛋白。随后，硝基酪氨酸-β-肌动蛋白聚合物与占地粘附的肌酸蛋白和/或卵线闭塞蛋白不复杂，足以维持屏障功能。硝基酪氨酸β-肌动蛋白的形成是由于皮质β-肌动蛋白附近的Onoo_生成增加。 onoo_是由.o2_的反应产生的，这分别是在周围膜上激活的NADPH-氧化酶和eNOS。 The activation of NADPH-oxidase is because of PKC" dependent phosphorylation of p47 phox. A novel mechanism for the increases in endothelial permeability in response to TNF will be investigated in this proposal. The Specific Objectives are: (1) To determine if PKCalpha-induced NADPH-oxidase activation mediates the ONOO_-induced change in nitrated-actin and actin肺部微血管内皮细胞单层（PMEM）的形态（PMEM）响应TNF（YEARL-2）（2），以确定肌动蛋白的酪氨酸残基允许肌动蛋白形态的变化和对TNF响应的内皮渗透性的变化（2-3年）。内皮渗透性是由于β-肌动蛋白与带状粘附的肌动蛋白结合蛋白的变化而变化（分别为-Catenin和ZO-1）（分别为3-4岁）。