Molecular Mechanisms in Abdominal Aortic Aneuysm

摘要 在前一项资助的支持下，我们检验了重要的压力调节因子PKCδ， 在动脉瘤发展过程中，会导致血管炎症和血管细胞耗尽。 使用遗传学、分子和药理学方法的组合，我们展示了 蛋白激酶Cδ在调节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和坏死性下垂之间的新联系，这是一种首次描述的程序性坏死 十年前。受体相互作用蛋白激酶3(RIP3)及其伴侣RIP1是为数不多的已被鉴定的基因之一 坏死性上睑下垂的中介物，强调了在这一途径中定位蛋白激酶Cδ的重要性。我们 结果表明，在人的动脉瘤组织中，PKCδ和RIP3的SMC水平均上调。在老鼠身上， PKCδ(Prkcd-/-)或Rip3(Rip3-/-)基因缺失产生与动脉瘤抵抗相关的表型 SMC保存完好，炎症减轻。在初步研究中，我们发现pkcδ调控rip3。 主动脉SMCs中的基因转录，这一新发现有望促进RIP3生物学的发展。健壮的 RIP3-/-和RIP3+/-的动脉瘤保护表型引发的化学文库筛选导致了 发现一类高效安全的RIP3抑制剂。在此续订申请中，我们假设PKCδ- STAT3信号是SMC RIP3表达决定因素，抑制RIP3可能会减弱 先前存在的动脉瘤的生长。我们未来5年的目标包括1)确定分子 PKCδ介导RIP3基因在主动脉瘤壁中表达的机制，2)建立 3)使用新的RIP3抑制剂阻断 先前存在的小鼠动脉瘤的疾病进展。提出了两个独立的具体目标。具体而言 目的1，我们计划通过拯救pRKCD-/-SMC来证明STAT3在PKCδ‘RIP3的S调控中的关键作用。 体质活跃的状态3.接下来，我们将确定STAT3 Serine727，一个研究较少的调控基因 机制，是通过蛋白激酶Cδ依赖的机制在动脉瘤组织中磷酸化。从机械上讲，我们 假设STAT3通过下游顺式元件调控RIP3基因转录。我们将对此进行测试 假设使用了尖端技术，如芯片测序和CRISPR/Cas9介导的基因编辑。 具体目标2既是基础性的，也是转化性的，目的是解决目前在坏死性下垂方面的知识空白 生物学和推进动脉瘤的治疗发展。我们将利用新的RIP3抑制剂来研究 坏死性下垂，解决RIP3激酶抑制和诱导细胞凋亡的关系并鉴定 专用于SMC的新RIP3底物。除了假设驱动的方法外，我们还将使用磷化- 蛋白质组学，以无偏见地识别SMC特有的坏死性下垂途径的新成分。最后，我们会 在体内证明新的RIP3抑制剂可以逆转SMC耗竭、组织破坏和炎症 给已经存在动脉瘤扩张的小鼠注射。建议研究提供的资料如下： 可能对程序性坏死和动脉瘤治疗的理解产生很大影响 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.