DAMP-RAGE Signaling and Fetal Injury in Inflammation-Induced Preterm Birth

描述(由申请人提供)：宫内和/或胎儿炎症、早产和包括脑性瘫痪在内的不良新生儿结局的发生率之间存在很强的相关性。如果要开发有针对性的干预措施来改善预后，我们需要充分了解无意中导致胎儿细胞损伤和组织损伤的分子机制。损伤相关分子模式分子(DAMP)是一组细胞内多效性蛋白，包括高迁移率族蛋白1(HMGB1)和S100钙结合蛋白家族成员(即S100 A12、S100A8、S100B)。当由于炎症或氧化应激而释放到细胞外腔时，通过激活内源性受体，如晚期糖基化终末产物受体(RAGE)，湿气就成为“危险信号”。RAGE的参与导致细胞功能障碍和损伤，由氧化应激和核因子-kappaB(NF：B)的持续活性驱动。利用蛋白质组学，我们发现S100A12通过激活RAGE在协调羊膜内炎性反应中发挥关键作用。我们还表明，具有高度炎症状态的人类胎儿体内的原型阻滞剂水平增加，如HMGB1和S100B。此外，在内毒素导致胎儿损伤的小鼠模型中，我们证明了母体炎症与胎儿氧化应激和细胞内抗氧化剂谷胱甘肽的耗竭有关。在同一实验模型中，我们进一步提供了HMGB1和RAGE在宫内炎症暴露的胎儿肝脏和脑中过表达的证据。这一知识体系与HMGB1和S100蛋白是假定的RAGE配体的证据相印证，使我们提出了在羊膜内感染和早产的背景下，阻滞轴在诱导产前终末器官损伤中的积极作用。为了验证这一假说，将追求三个目标：1)特定目标1旨在提供体内观察证据，证明子宫内炎症导致人类胎儿氧化还原稳态的失衡，导致潮湿蛋白的释放，并通过RAGE激活导致产前胎儿细胞损伤；2)在特定目标2中，我们将在体外探索典型的DAMPS和RAGE信号在诱导细胞损伤中的机制和功能作用。我们将测试抗HMGB1和抗RAGE阻断抗体或潜在的治疗药物如N-乙酰半胱氨酸和丙酮酸乙酯逆转湿气轴激活的损伤效应的有效性；3)在特定目标3，通过使用独特的基因工程RAGE缺陷(RAGE-/-)和RAGE转基因(RAGETg+)动物，我们计划深入了解RAGE信号作为导致胎儿损伤的常见和必需途径，并探索潮湿和RAGE拮抗作为体内治疗策略的价值。通过完成这些目标，我们希望对导致早产儿终末器官损伤的过程提供新的见解，并识别能够调节胎儿炎症反应并改善结局的新的分子靶点。 公共卫生相关性：损伤相关分子模式分子(DAMP)和晚期糖基化终末产物受体(RAGE)是细胞损伤的内源性介质，导致不可逆的组织损伤。我们发现，在合并羊膜内感染的妊娠中，炎症和氧化应激的高度状态具有触发重要胎儿器官RAGE参与的所有必要属性，这可能解释了与早产相关的不良结局发生率增加的原因。通过使用基因工程动物模型，我们的目标是深入了解胎儿RAGE激活的病理后果，以及RAGE靶向治疗干预降低早产儿新生儿发病率和死亡率的潜力。

DESCRIPTION (provided by applicant): Strong correlations exist between intra-uterine and/or fetal inflammation, prematurity and incidence of adverse neonatal outcomes, including cerebral palsy. If targeted interventions to improve outcome are to be developed we need to fully understand the molecular mechanisms which inadvertently cause fetal cell damage and tissue injury. Damage-associated molecular pattern molecules (DAMPs) are a pleiotropic group of intra-cellular proteins including high-mobility group box 1 protein (HMGB1) and members of the S100 calcium binding protein family (i.e., S100 A12, S100A8, S100B). When released into the extra-cellular compartment as a result of inflammation or oxidative stress, DAMPs become "danger signals" by activating endogenous receptors such as the receptor of advanced glycation end-products (RAGE). Engagement of RAGE leads to cellular dysfunction and injury driven by oxidative stress and sustained activity of nuclear factor-kappa B (NF:B). Using proteomics we discovered that S100A12 plays a key role in orchestrating the intra-amniotic inflammatory response to infection via RAGE activation. We have also shown that human fetuses with heightened inflammatory statuses have increased systemic levels of prototype DAMPs such as HMGB1 and S100B. In addition, in a mouse model of endotoxin induced fetal damage we demonstrated that maternal inflammation is associated with fetal oxidative stress and depletion of the intracellular antioxidant glutathione. In the same experimental model we further provided evidence that HMGB1 and RAGE and over-expressed in the liver and brain of the fetuses exposed to inflammation in utero. This body of knowledge, corroborated with the evidence that HMGB1 and S100 proteins are putative RAGE ligands, has led us to propose an active role for the DAMP- RAGE axis in inducing antenatal end-organ damage in the setting of intra-amniotic infection and prematurity. To test this hypothesis, three aims will be pursued: 1) Specific Aim 1 is geared to provide in vivo observational evidence that intra-uterine inflammation induces an imbalance in the redox homeostasis of the human fetus, causing release of DAMP proteins and antenatal fetal cellular damage via RAGE activation; 2) In Specific Aim 2 we will explore, in vitro, the mechanism and functional role of prototypical DAMPs and RAGE signaling in inducing cellular injury in a relevant bioassay. We will test the efficacy of anti-HMGB1 and anti-RAGE blocking antibodies or potential therapeutic agents such as N-acetylcysteine and ethyl pyruvate to reverse the damaging effects of the DAMP-RAGE axis activation; 3) In Specific Aim 3, by using unique genetically engineered RAGE deficient (RAGE-/-) and RAGE transgenic (RAGETg+) animals, we plan to provide insight into RAGE signaling as the common and obligatory pathway leading to fetal damage and explore the value of DAMP and RAGE antagonism as treatment strategies in vivo. By completing these aims we hope to provide novel insight into the processes leading to end-organ damage in premature infants and identify new classes of molecular targets that can modulate the inflammatory response of the fetus and improve outcomes. PUBLIC HEALTH RELEVANCE: Damage-associated molecular pattern molecules (DAMPs) and the receptor of advanced glycation end- products (RAGE) are endogenous mediators of cellular injury leading to irreversible tissue damage. We discovered that in pregnancies complicated by intra-amniotic infection, the heightened states of inflammation and oxidative stress have all the requisite attributes to trigger RAGE engagement in vital fetal organs that may explain the increased incidence of poor outcomes associated with prematurity. By using genetically engineered animal models, we aim to provide insight into the pathological consequences of fetal RAGE activation and the potential of RAGE targeted therapeutic interventions to lower the neonatal morbidity and mortality of premature infants.