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

描述（由申请人提供）：子宫内和/或胎儿炎症、早产与新生儿不良结局（包括脑瘫）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以改善结果，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（DAMPs）是一组多向性细胞内蛋白，包括高迁移率组盒1蛋白（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时，DAMPs通过激活内源性受体（如晚期糖基化终产物受体（RAGE））成为“危险信号”。RAGE的参与导致氧化应激和核因子- κ B （NF:B）持续活性驱动的细胞功能障碍和损伤。利用蛋白质组学，我们发现S100A12通过RAGE激活在协调羊膜内炎症反应中发挥关键作用。我们还表明，炎症状态升高的人类胎儿会增加原型DAMPs（如HMGB1和S100B）的全身水平。此外，在内毒素诱导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消耗有关。在相同的实验模型中，我们进一步提供了HMGB1和RAGE在子宫内暴露于炎症的胎儿的肝脏和大脑中过表达的证据。这一知识体系与HMGB1和S100蛋白被认为是RAGE配体的证据相证实，使我们提出了在羊膜内感染和早产的情况下，DAMP- RAGE轴在诱导胎儿终末器官损伤中的积极作用。为了验证这一假设，将追求三个目标：1)特异性目标1旨在提供体内观察证据，证明子宫内炎症诱导人类胎儿氧化还原稳态失衡，通过RAGE激活导致DAMP蛋白释放和产前胎儿细胞损伤；2)在Specific Aim 2中，我们将通过相关的生物测定，在体外探索原型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n -乙酰半胱氨酸和丙酮酸乙酯等潜在治疗药物的疗效，以逆转DAMP-RAGE轴激活的破坏性影响；3)在Specific Aim 3中，我们计划通过独特的基因工程RAGE缺陷（RAGE-/-）和RAGE转基因（RAGETg+）动物，深入了解RAGE信号作为导致胎儿损伤的共同和强制性途径，并探索DAMP和RAGE拮抗剂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，并确定可以调节胎儿炎症反应并改善结果的新分子靶点。

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.