Endothelial Dysfunction and Vascular Inflammation

介绍和目的：脓毒性休克标志着严重感染时级联反应压倒代偿机制，导致明显的心血管衰竭。需要低血压的血管加压药的出现大大增加了感染死亡的风险。高达60%的败血性休克非幸存者在疾病的前7-10天死于难治性休克。在难治性感染性休克中，血管舒张和收缩最终受损，急性异常类似于慢性动脉粥样硬化中的内皮功能障碍和损伤。 本研究旨在探讨内皮功能障碍和血管炎症的介质、信号转导途径和潜在机制。 建议的工作路线： 探索一氧化氮触发的信号转导通路在人-小鼠杂交内皮细胞系和原代人内皮细胞。初步研究表明，外源性一氧化氮抑制蛋白酶体功能并激活p38 MAPK。其他工作已经证明了一氧化氮信号传导和过氧化物酶体增殖物激活受体（PPARs）之间潜在的重要相互作用，如一氧化氮，与内皮保护和血管健康的其他方面有关。发现一氧化氮激活PPARgamma，从而调节含有PPAR-response元件的下游靶基因。 研究NOS抑制剂和TNF α刺激的人原代肺微血管内皮细胞炎症反应之间的相互作用。 在原代细胞中使用RNA介导的干扰（RNAi）方法开发内皮细胞功能障碍的体外模型。内皮功能障碍与包括脓毒症和动脉粥样硬化在内的多种模型和临床环境中eNOS表达或功能降低相关。基因敲除后，在存在和不存在炎症介质激活（TNF α）的情况下，使用蛋白质印迹、流式细胞术和寡核苷酸微阵列进行表型表征。 在平行实验中，RNAi将用于敲低BMPR 2基因。BMPR 2功能的丧失与原发性和继发性肺动脉高压有关，这是一种影响肺血管的内皮功能障碍形式。目前，计划将联合收割机BMPR 2敲除与eNOS敲除以2 × 2设计组合，随后进行表型表征和表达谱分析。将在方案（与Michael所罗门，医学博士）的临床样本背景下检查和分析来自该体外工作的数据。招募原发性肺动脉高压患者。 进度： 用人eNOS转染单胚U937细胞产生了一种细胞系，该细胞系响应钙离子载体而产生一氧化氮，但在静息状态下不产生（Blood，1997）。然而，在分化佛波醇-12-乙酸酯-13-肉豆蔻酸酯后，eNOS表达细胞通过独立于一氧化氮的机制产生增加量的TNF α和活性氧。 NOS抑制剂N-甲基-L-精氨酸和eNOS L-精氨酸结合位点的突变均不能阻断eNOS上调TNF α的能力。相反，与超氧化物歧化酶共转染或eNOS的NADPH结合位点的缺失完全阻止eNOS上调TNF α的产生。这些结果表明，eNOS可以通过一氧化氮（J Immunol，1994; J Biol Chem，1997）和基于活性氧的信号转导途径（J Biol Chem，2000）来调节炎症反应。由eNOS产生的超氧化物显示通过p42/44 MAPK活化上调TNF α（J Biol Chem，2001）。

Introduction and Objective: Septic shock marks the point in a severe infection when cascading responses overwhelm compensatory mechanisms resulting in overt cardiovascular failure. The appearance of vasopressor requiring hypotension substantially increases the risk of death from infection. Up to 60% of septic shock non-survivors die in refractory shock during the first 7-10 days of illness. In refractory septic shock both vascular relaxation and constriction ultimately become impaired, acute abnormalities analogous to endothelial dysfunction and injury in chronic atherosclerosis. This investigation is exploring the mediators, signal transduction pathways, and underlying mechanisms of endothelial dysfunction and vascular inflammation. Proposed Course of Work: Explore nitric oxide-triggered signal transduction pathways in a human-mouse hybrid endothelial cell line and primary human endothelial cells. Initial studies demonstrate that exogenous nitric oxide inhibits proteosame function and activates p38 MAPK. Other work has demonstrated potentially important interactions between nitric oxide signaling and peroxisome proliferator-activated receptors (PPARs), like nitric oxide, have been associated with endothelial protection and other aspects of vascular health. Nitric oxide was found to activate PPARgamma and thereby regulate downstream target genes that contain PPAR-response elements. Investigate interactions between NOS inhibitors and TNF alpha-stimulated inflammatory responses in human primary pulmonary microvascular endothelial cells. Develop an in vitro model of endothelial cell dysfunction using a RNA-mediated interference (RNAi) approach in primary cells. Endothelial dysfunction has been associated with reduced eNOS expression or function in a wide variety of models and clinical settings including sepsis and atherosclerosis. Gene knockdown will be followed by phenotypic characterization using Western blot, flow cytometry, and oligonucleotide microarrays in the presence and absence of inflammatory-mediator activation (TNFalpha). In parallel experiments, RNAi will be used to knockdown the BMPR2 gene. Loss of BMPR2 function has been linked to primary and secondary pulmonary hypertension, a form of endothelial dysfunction that affects the pulmonary vasculature. At present, it is planned to combine BMPR2 knockdown with eNOS knockdown in a 2 x 2 design followed by phenotypic characterization and expression profiling. Data from this in vitro work will be examined and analyzed in the context of clinical samples from a protocol (with Michael Solomon, M.D.) enrolling patients with primary pulmonary hypertension. Progress: Transfection of monoblastoid U937 cells with human eNOS resulted in a cell line that produced nitric oxide in response to calcium ionophore, but not in the resting state (Blood, 1997). However, after differentiation with phorbol-12-acetate-13-myristate, eNOS expressing cells produced increased amounts of both TNFalpha and reactive oxygen species by mechanisms that were independent of nitric oxide. Neither Nw-methyl-L-arginine, a NOS inhibitor, nor mutation of the L-arginine binding site of eNOS, rendering it incapable of producing nitric oxide, blocked the ability of eNOS to upregulate TNFalpha. Conversely, co-transfection with superoxide dismutare or deletion of the NADPH binding site of eNOS completely prevented eNOS from upregulating TNFalpha production. These results suggested that eNOS can regulate inflammatory responses through both nitric oxide (J Immunol, 1994; J Biol Chem, 1997) and reactive oxygen species-based signal transduction pathways (J Biol Chem, 2000). Superoxide produced by eNOS was shown to upregulate TNFalpha via p42/44 MAPK activation (J Biol Chem, 2001).