Molecular Mechanisms in Abdominal Aortic Aneuysm

摘要 在之前资助的支持下，我们测试了PKCδ（一种重要的压力调节因子）的假设， 在动脉瘤发展过程中，导致平滑肌细胞耗竭和血管炎症。 通过遗传学、分子学和药理学方法的结合，我们证明了 PKCδ在动脉瘤血管平滑肌细胞凋亡中的作用没想到，我们也 发现了PKCδ和坏死性凋亡之间的一种新的联系， 十几年前受体相互作用蛋白激酶3（RIP 3）及其伴侣RIP 1是少数被鉴定的 PKC δ是坏死性凋亡的重要介质，强调了PKCδ在该通路中的重要性。我们 证明在人类动脉瘤组织中，SMC中PKCδ和RIP 3的水平均上调。在小鼠中， PKCδ（Prkcd-/-）或RIP 3（Rip 3-/-）基因缺失产生了与细胞周期相关的耐药表型。 平滑肌细胞得以保存炎症也有所减轻在初步研究中，我们发现PKCδ调节Rip 3， 主动脉SMC中的基因转录，这是一项新的发现，有望推动RIP 3生物学的发展。鲁棒 Rip 3-/-和Rip 3 +/-的细胞周期保护表型激发了化学文库筛选，其导致了Rip 3-/-和Rip 3 +/-的表达。 发现了一类有效且安全的RIP 3抑制剂。在这项更新申请中，我们假设PKCδ- STAT 3信号传导是SMC Rip 3表达的决定因素，Rip 3的抑制可能会减弱 既存动脉瘤的生长。我们未来5年的目标包括：1）确定 PKCδ介导的Rip 3基因在动脉瘤壁表达的机制; 2）建立一个更完整的 在主动脉SMC中全面的坏死性凋亡信号网络，和3）使用新的RIP 3抑制剂阻断 小鼠中预先存在的动脉瘤的疾病进展。提出了两个独立的具体目标。在特定 目的1，我们计划证明STAT 3在PKCδ调控Rip 3中的关键作用，通过用一种新的方法来拯救Prkcd-/- SMCs。 组成型活性STAT 3。接下来，我们将确定STAT 3丝氨酸727，一个研究较少的调节蛋白， 在动脉瘤组织中通过PKCδ依赖性机制磷酸化。机械地，我们 推测STAT 3通过下游顺式元件调控Rip 3基因的转录。我们将测试这个 利用芯片测序和CRISPR/Cas9介导的基因编辑等尖端技术进行假设。 具体目标2是基础和翻译，其目标是解决目前在坏死性凋亡方面的知识空白 生物学和推进动脉瘤的治疗发展。我们将利用新的RIP 3抑制剂来研究 坏死性凋亡，解决RIP 3激酶抑制和细胞凋亡诱导之间的关系，并确定 新的SMC特异性RIP 3底物。除了假设驱动的方法，我们将采用磷酸- 蛋白质组学无偏地鉴定SMC特有的坏死性凋亡途径的新组分。最后我们将 在体内证明，新的RIP 3抑制剂可以逆转SMC消耗，组织破坏和炎症， 给预先存在动脉瘤扩张的小鼠施用。拟议研究所提供的资料如下： 可能会对程序性坏死的理解以及动脉瘤治疗产生很大影响 发展

ABSTRACT Under the support of the previous grant, we tested the hypothesis that PKCδ, an important stress regulator, contributes to smooth muscle cell depletion and vascular inflammation during aneurysm development. Using a combination of genetic, molecular, and pharmacological approaches, we demonstrated the essential role of PKCδ in regulating vascular smooth muscle cell (SMC) apoptosis in aneurysm. Unexpectedly, we also discovered a novel connection between PKCδ and necroptosis, a form of programmed necrosis first described a decade ago. Receptor-interacting protein kinase 3 (RIP3) and its partner RIP1 are among the few identified mediators of necroptosis, underscoring the significance of positioning PKCδ within this pathway. We demonstrated that in human aneurysm tissue, SMC levels of both PKCδ and RIP3 was upregulated. In mice, gene deletion of either PKCδ (Prkcd-/-) or RIP3 (Rip3-/-) produced an aneurysm-resistant phenotype associated with preserved SMCs and diminished inflammation. In preliminary studies, we showed that PKCδ regulates Rip3 gene transcription in aortic SMCs, a novel finding highly expected to advance RIP3 biology. The robust aneurysm-protective phenotype of Rip3-/- and Rip3+/- motivated a chemical library screening which led to the discovery of a class of potent and safe RIP3 inhibitors. In this renewal application, we hypothesize that PKCδ- STAT3 signaling is a determinant of SMC Rip3 expression and that inhibition of RIP3 may attenuate growth of pre-existing aneurysms. Our objectives for the next 5 years include 1) to determine the molecular mechanism underlying PKCδ-mediated Rip3 gene expression in aneurysmal aortic wall, 2) to establish a more comprehensive necroptosis signaling network in aortic SMCs, and 3) to use the new RIP3 inhibitor to block disease progression of pre-existing aneurysms in mice. Two independent specific aims are proposed. In Specific Aim 1, we plan to prove the critical role of STAT3 in PKCδ's regulation of Rip3 by rescuing Prkcd-/- SMCs with a constitutively active STAT3. Next, we will determine whether STAT3 Serine727, a less studied regulatory mechanism, is phosphorylated in aneurysm tissue via a PKCδ-dependent mechanism. Mechanistically, we postulate that STAT3 regulates Rip3 gene transcription through a downstream cis-element. We will test this hypothesis using cutting edge technologies such as Chip-sequencing and CRISPR/Cas9-mediated gene editing. Specific Aim 2 is both basic and translational, with a goal to address the current knowledge gaps in necroptosis biology and to advance therapeutic development for aneurysm. We will utilize the new RIP3 inhibitors to study necroptosis, addressing the relationship between RIP3 kinase inhibition and apoptosis induction and identifying new RIP3 substrates specific to SMCs. In addition to hypothesis-driven approaches, we will employ phospho- proteomics to unbiasedly identify new components of the necroptosis pathway unique to SMCs. Finally, we will prove in vivo that the new RIP3 inhibitors can reverse SMC depletion, tissue destruction and inflammation when administered to mice with pre-existing aneurysmal dilations. Information produced by the proposed studies is likely to have a high impact on the understanding of programmed necrosis as well as aneurysm therapeutic development.