Supraphysiologic Shear Stresses Associated with Cardiopulmonary Bypass are Sufficient to Activate RIKP3 Signaling

绝大多数儿科心内直视手术都要求病人有心肺支持。 搭桥(CPB)。暴露在CPB中会导致全身炎症和由此导致的多器官功能障碍。CPB后 炎症被认为是由于患者的血细胞暴露在CPB的塑料管中而引起的 电路和非生理性的高剪应力。然而，这一过程背后的机制尚不清楚。 我们的长期目标是了解这些侮辱是如何导致CPB术后炎症的，并将其转化为 将知识转化为新的治疗策略。这个项目的科学前提是受体相互作用 丝氨酸/苏氨酸蛋白激酶3(RIPK3)信号是体外循环相关炎症的一种新的介质 可以有针对性地改善新生儿心脏手术患者的预后。新生儿CBP患者的数据，a CPB的大动物模型和体外实验构成了我们的科学前提基础。我们最近 报道称，RIPK3和坏死性下垂在体外循环术后炎症中起作用。我们的数据表明，RIPK3 在体外循环术后炎症中起作用。我们确定CPB期间存在超生理剪应力 足以在体外和体外循环仔猪模型中激活RIPK3信号。从机制上讲，我们发现 剪切力启动的钙信号通路对单核细胞的激活至关重要。我们有 确定了可以通过小分子靶向减少CPB激活RIPK3的特定途径 减少全身炎症反应和器官功能障碍的目标。我们假设与CPB相关的 剪切力激活RIPK3介导的炎症。这项提案的目标是1)确定如何 CPB激活RIPK3信号，2)阐明RIPK3信号如何参与CPB相关 炎症/器官功能障碍，以及3)确定阻断RIPK3信号是否足以减少CPB- 相关的炎症和器官功能障碍。我们的方法将包括两个具体目标： 目的1.确定切应力如何激活循环髓系细胞中的RIPK3信号。 我们假设超生理剪应力足以激活RIPK3信号。我们将描述 有灶体外循环中RIPK3激活的剪应力阈值及其分子机制 关于细胞皮质、钙信号级联和剪切反应蛋白在这一反应中所起的作用。 目的2.证明RIPK3信号介导体外循环相关的炎症和器官功能障碍。 我们推测RIPK3信号是CPB炎症反应所必需的。RIPK3可以帮助传播 通过坏死性下垂、细胞因子的释放和白细胞的迁移引起的炎症。我们将在体外和在 体内实验证明，靶向RIPK3信号可以减少CPB相关的炎症。 这项研究是新的和有意义的-阐明CPB如何激活RIPK3信号和坏死性下垂可能 启用CPB患者的新治疗模式，改善结果，并降低医疗成本，因为 所提出的信号通路可以在临床使用或临床前开发中被小分子靶向。

The vast majority of pediatric open-heart surgeries require the patient to be supported by cardiopulmonary bypass (CPB). Exposure to CPB causes systemic inflammation and resultant multi-organ dysfunction. Post-CPB inflammation is believed to be caused by exposure of the patient’s blood cells to the plastic tubing of the CPB circuit and unphysiological high shear stress. However, the mechanisms underlying this process are unclear. Our long-term goal is to understand how these insults contribute to post-CPB inflammation and translate this knowledge into novel treatment strategies. The scientific premise for this project is that Receptor-Interacting serine/threonine-Protein Kinase 3 (RIPK3) signaling is a novel mediator of CPB associated inflammation that can be targeted to improve outcomes for neonatal cardiac surgery patients. Data from neonatal CBP patients, a large animal model of CPB, and in vitro experiments form the basis of our scientific premise. We recently published that RIPK3 and necroptosis play a role in post-CPB inflammation. Our data demonstrate that RIPK3 plays a role in post-CPB inflammation. We established that supraphysiologic shear stresses present during CPB are sufficient to activate RIPK3 signaling in vitro and in a piglet model of CPB. Mechanistically, we found that shear stress-initiated calcium signaling pathways are critical to the activation of monocytic cells. We have identified specific pathways that can be targeted with small molecules to reduce CPB-activation of RIPK3 with goal of reducing systemic inflammatory response and organ dysfunction. We hypothesize that CPB-associated shear stress activates RIPK3 mediated inflammation. The objectives of this proposal are 1) to determine how CPB activates RIPK3 signaling, 2) elucidate how RIPK3 signaling contributes to CPB-associated inflammation/organ dysfunction, and 3) determine if blocking RIPK3 signaling is sufficient to reduce CPB- associated inflammation and organ dysfunction. Our approach will consist of two specific aims: Aim 1. Determine how shear stress activates RIPK3 signaling in circulating myeloid cells. We postulate that supraphysiologic shear stress is sufficient to activate RIPK3 signaling. We will characterize the shear stress thresholds and molecular mechanism responsible for RIPK3 activation during CPB with a focus on the roles that the cell cortex, calcium signaling cascade, and shear responsive kinases play in this response. Aim 2. Demonstrate that RIPK3 signaling mediates CPB-associated inflammation and organ dysfunction. We postulate that RIPK3 signaling is required for the inflammatory response to CPB. RIPK3 can help propagate inflammation via necroptosis, the release of cytokines, and leukocyte migration. We will perform in vitro and in vivo experiments to demonstrate that targeting RIPK3 signaling reduces CPB-associated inflammation. This research is novel and significant – elucidating how CPB activates RIPK3 signaling and necroptosis could enable a new treatment paradigm for CPB patients, improve outcomes, and reduce healthcare costs, since the proposed signaling pathways can be targeted by small molecules in clinical use or pre-clinical development.