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、早产和新生儿不良结局（包括脑瘫）发生率之间存在强相关性。如果有针对性的干预措施，以改善结果的发展，我们需要充分了解无意中造成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）是一组多效性的细胞内蛋白，包括高迁移率族蛋白1（HMGB 1）和S100钙结合蛋白家族的成员（即，S100 A12、S100 A8、S100 B）。当由于炎症或氧化应激而释放到细胞外隔室中时，DAMP通过激活内源性受体如晚期糖基化终产物受体（AGEs）而成为“危险信号”。参与的NF导致细胞功能障碍和损伤驱动的氧化应激和核因子-κ B（NF：B）的持续活性。使用蛋白质组学，我们发现S100 A12在通过活化的羊膜内炎症反应中起着关键作用。我们还发现，炎症状态升高的人类胎儿体内原型DAMP（如HMGB 1和S100 B）的水平也有所增加。此外，在内毒素诱导的胎儿损伤的小鼠模型中，我们证明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，我们进一步提供了证据表明HMGB 1和HMGB 2在子宫内暴露于炎症的胎儿的肝脏和大脑中过表达。这一知识体系，与HMGB 1和S100蛋白是推定的DAP 100配体的证据相证实，使我们提出DAMP-DAP 100轴在羊膜内感染和早产的情况下诱导产前终末器官损伤中的积极作用。1）具体目标1旨在提供体内观察证据，表明子宫内炎症诱导人胎儿氧化还原稳态失衡，导致DAMP蛋白质释放和产前胎儿细胞损伤（通过β-内酰胺酶激活）; 2）在具体目标2中，我们将在体外探索原型DAMP和DAMP信号在相关生物测定中诱导细胞损伤的机制和功能作用。我们将测试抗HMGB 1和抗HMGB 2阻断抗体或潜在治疗剂如N-乙酰半胱氨酸和丙酮酸乙酯逆转DAMP-HMGB 1轴激活的损伤作用的功效; 3）在具体目标3中，通过使用独特的遗传工程改造的RAGETg缺陷（RAGETg-/-）和RAGETg转基因（RAGETg+）动物，我们计划提供对导致胎儿损伤的共同和强制性途径的cAMP信号传导的深入了解，并探索DAMP和cAMP拮抗作用作为体内治疗策略的价值。通过完成这些目标，我们希望为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局的新分子靶点。 公共卫生相关性：损伤相关分子模式分子（DAMP）和晚期糖基化终产物受体（DAMP）是导致不可逆组织损伤的细胞损伤的内源性介质。我们发现，在羊膜内感染并发的妊娠中，炎症和氧化应激状态的升高具有触发重要胎儿器官的炎症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。通过使用基因工程动物模型，我们的目标是提供洞察胎儿胎盘激活的病理后果和潜在的胎盘靶向治疗干预，以降低新生儿发病率和早产儿死亡率。

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.