Targeting Pathogenic Endothelial Dysfunction in Systemic Lupus Erythematosus

 描述（由申请人提供）： 狼疮性肾炎（LN）影响所有系统性红斑狼疮（SLE）患者的一半。在非洲裔美国妇女中，退伍军人人口中的人口不断增长，这种疾病导致五年内50%的肾衰竭率。导致LN的异质性机制阻碍了有效的LN药物开发。内皮细胞通过内皮型一氧化氮合酶（endothelial nitric oxide synthase，eNOS）和一氧化氮（nitric oxide，NO）的作用，对多种炎症反应起最终的共同损伤作用。狼疮的特征在于这些细胞的功能障碍（内皮细胞功能障碍或ECD）。这种功能障碍导致组织炎症，如增生性狼疮性肾炎。了解导致ECD的机制对于开发新的和更广泛有效的LN治疗方法至关重要。初步研究表明，eNOS在苏氨酸495（Thr-495）的异常磷酸化和eNOS同源二聚体的解偶联导致SLE中的ECD。该建议的目标是定义和靶向导致功能性ECD的常见细胞内机制，并在发现分析中确定LN中ECD信号的血清因子。为了实现这一目标，提出了以下目标：1）表征SLE和LN ECD血清诱导的eNOS功能障碍的细胞内途径。我们推测SLE血清通过Thr-495磷酸化增加和eNOS二聚体解偶联的共同最终细胞内机制诱导ECD。为了解决这一假设，将对来自SLE、LN和健康对照患者的血清样品诱导中性粒细胞迁移和粘附至培养的EC（体外ECD）的能力进行表型分析。用来自这些组的血清培养后，将测定eNOS和磷酸化eNOS蛋白表达和二聚体偶联。2)评估靶向eNOS功能障碍途径的药物对SLE血清诱导的体外ECD的影响。我们假设，由SLE血清诱导的ECD可以被逆转与设计用于恢复eNOS偶联，抑制PKC介导的Thr-495磷酸eNOS或模拟eNOS NO的药物。这一目的将确定这些方法中的每一种恢复内皮功能的能力与ECD诱导血清培养的细胞。将靶向基于机制的ECD的试剂加入到具有ECD诱导SLE血清的培养物中，以测试对ECD的影响。药效学标志物将用于确定每种药物的最佳浓度和基于机制的作用，以及对EC功能的预期作用。3)在ECD血清中具有差异表达的血清蛋白和外泌体脂质的发现分析。初步研究表明，狼疮免疫球蛋白组分和外来体诱导ECD。初步发现分析表明，外泌体中的鞘糖脂与对治疗的不良反应相关。将ECD和非ECD LN、SLE和对照血清分级分离成外泌体、免疫球蛋白和非免疫球蛋白、非外泌体级分，并测试体外ECD诱导。诱导ECD的上述组分特有的蛋白质将通过液相色谱法和飞行时间/飞行时间质谱法测定。将通过液相色谱法和质谱法测定ECD血清特有的外来体脂质。途径分析将提示从ECD组分鉴定的分子之间的ECD途径。这个项目将填补我们对ECD如何诱导SLE病理性炎症浸润的知识空白，并加速SLE退伍军人的靶向治疗。这项研究可能会加速发展的ECD疾病，如高血压，动脉粥样硬化，糖尿病肾病的退伍军人的治疗。

 DESCRIPTION (provided by applicant): Lupus nephritis (LN) affects half of all systemic lupus erythematosus (SLE) patients. Among African-American women, a growing demographic in the Veteran population, this disease leads to 50% renal failure rate by five years. Effective LN drug development is hindered by the heterogeneity mechanisms leading to LN. Endothelial cells act as a final common dampener on inflammatory responses of many types through the action of endothelial nitric oxide synthase (eNOS) nitric oxide (NO). Lupus is characterized by dysfunction of these cells (endothelial cell dysfunction or ECD). This dysfunction leads to tissue inflammation such as proliferative lupus nephritis. Knowledge of mechanisms leading to ECD is essential to progress in developing novel and more broadly effective therapies for LN. Preliminary studies suggest that abnormal phosphorylation of eNOS at threonine 495 (Thr-495) and uncoupling of eNOS homodimers leads to ECD in SLE. The goal of this proposal is to define and target the common intracellular mechanisms leading to functional ECD and determine, in a discovery analysis, serum factors that signal for ECD in LN. To achieve this goal, the following aims are proposed: 1) Characterize intracellular pathways of eNOS dysfunction induced by SLE and LN ECD serum. We hypothesize that SLE serum induces ECD through the common final intracellular mechanisms of increased Thr-495 phosphorylation and eNOS dimer uncoupling. To address this hypothesis, serum samples from SLE, LN, and healthy control patients will be phenotyped for their ability to induce neutrophil migration and adhesion to cultured EC (in vitro ECD). eNOS and phospho eNOS protein expression and dimer coupling will be determined after culture with serum from these groups. 2) Assess the effect of agents that target pathways of eNOS dysfunction on SLE serum-induced in vitro ECD. We hypothesize that ECD induced by SLE serum can be reversed with agents designed to restore eNOS coupling, inhibit PKC-mediated Thr-495 phospho-eNOS or mimic eNOS NO. This aim will determine the ability of each of these approaches to restore endothelial function in cells cultured with ECD-inducing serum. Agents that target mechanism-based ECD will be added to culture with ECD-inducing SLE serum to test the effect on ECD. Pharmacodynamic markers will be used to determine optimal concentrations and mechanism-based action of each agent in association with the desired effect on EC function. 3) Discovery analysis of serum proteins and exosome lipids with differential expression in ECD serum. Preliminary studies suggest that lupus immunoglobulin fractions and exosomes induce ECD. Preliminary discovery analysis demonstrates that glycosphingolipids in exosomes associate with poor response to therapy. ECD and non-ECD LN, SLE, and control serum will be fractionated into exosome, immunoglobulin, and non-immunoglobulin, non-exosome fractions and tested for in vitro ECD- induction. Proteins unique to the above fractions that induce ECD will be determined by liquid chromatography and time-of-flight/time-of-flight mass spectrometry. Exosome lipids unique to ECD serum will be assayed by liquid chromatography and mass spectrometry. Pathway analysis will suggest pathways of ECD among molecules identified from ECD fractions. This project will fill gaps in our knowledge of how ECD induces pathologic inflammatory infiltrates in SLE and accelerate targeted therapies for Veterans with SLE. This study could accelerate development of therapies for diseases with ECD such as hypertension, atherosclerosis, and diabetic renal disease in Veterans.