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

绝大多数小儿心脏直视手术需要患者在心肺功能支持下进行。 旁路（CPB）。暴露于CPB引起全身炎症和由此产生的多器官功能障碍。cpb后 炎症被认为是由患者的血细胞暴露于CPB的塑料管引起的 电路和非生理高剪切应力。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损伤如何导致CPB后炎症，并将其转化为 新的治疗策略。这个项目的科学前提是， 丝氨酸/苏氨酸-蛋白激酶3（RIPK 3）信号传导是CPB相关炎症的新介质， 可以有针对性地改善新生儿心脏手术患者的结局。新生儿CBP患者的数据，a CPB的大型动物模型和体外实验构成了我们科学前提的基础。我们最近 发表了RIPK 3和坏死性凋亡在CPB后炎症中起作用。我们的数据表明，RIPK 3 在CPB后炎症中起作用。我们确定CPB期间存在超生理剪切应力， 足以在体外和CPB的小猪模型中激活RIPK 3信号传导。从机制上讲，我们发现， 剪切应力引发的钙信号传导途径对于单核细胞的活化是关键的。我们有 确定了可以用小分子靶向的特定途径，以减少RIPK 3的CPB激活， 目的是减少全身炎症反应和器官功能障碍。我们假设CPB相关的 剪切应力激活RIPK 3介导的炎症。本提案的目标是：（1）确定如何 CPB激活RIPK 3信号，2）阐明RIPK 3信号如何有助于CPB相关的 炎症/器官功能障碍，和3）确定阻断RIPK 3信号传导是否足以减少CPB-1。 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： 目标1。确定切应力如何激活循环髓样细胞中的RIPK 3信号传导。 我们假设超生理剪切应力足以激活RIPK 3信号。我们将描述 体外循环中RIPK 3激活的切应力阈值和分子机制， 细胞皮质、钙信号级联和剪切响应激酶在这种反应中的作用。 目标二。证明RIPK 3信号转导介导CPB相关炎症和器官功能障碍。 我们推测RIPK 3信号是CPB炎症反应所必需的。RIPK 3可以帮助传播 炎症通过坏死性凋亡、细胞因子的释放和白细胞迁移。我们将在体外和体内 体内实验证明靶向RIPK 3信号传导减少CPB相关炎症。 这项研究是新颖的和有意义的-阐明CPB如何激活RIPK 3信号传导和坏死性凋亡可以 为CPB患者提供新的治疗模式，改善结局，降低医疗成本，因为 在临床使用或临床前开发中，小分子可以靶向拟议的信号通路。

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.