Coronary artery disease locus 1p32.2 and miR92a-PPAP2B signaling in endothelial mechanobiology

项目摘要 动脉粥样硬化性疾病仍然是美国人发病率和死亡率的主要原因。 动脉粥样硬化好发于动脉曲度和分叉处等血管内皮细胞 细胞被局部扰动的血流激活。最近的研究提出了新的基因和以前的发现-- 在机械上导致动脉粥样硬化的预期生物学；然而，遗传前病变之间的相互作用 动脉粥样硬化的处置和血流介导的内皮功能仍有待阐明。 全基因组关联研究(GWAS)和随后的研究发现，磷脂酸- 磷酸酶2B型(PPAP2B，也称为LPP3或PLPP3)作为一种位于CHR 1p32.2的基因，显著地 影响冠状动脉疾病(CAD)的风险。此外，内皮细胞PPAP2B减少与 增加对冠心病的易感性。我们最近发现了一种新的机制，将扰动流动与减少联系起来 在内皮PPAP2B中，通过microRNA介导的机械转导途径：扰乱的流动被激活 内皮miR-92a进而通过抑制PPAP2B直接和间接抑制PPAP2B 转录因子KLF2激活PPAP2B；内皮PPAP2B减少导致增加 内皮细胞炎症和单层完整性受损。此外，以前的研究表明， 全身应用miR-92a抑制剂可抑制多种组织中miR-92a的表达 小鼠的动脉粥样硬化。尽管这些结果表明流动敏感的miR92a-PPAP2B途径 与动脉粥样硬化易感的内皮细胞表型有关，潜在的因果关系 内皮细胞miR92a-PPAP2B途径和体内动脉粥样硬化的发生尚不清楚。此外，该机制 PPAP2B中与CAD相关的因果变异体通过什么发挥作用及其在内皮细胞中的潜在作用 机械转导仍未被探索。 我们已经收集了非常有力的证据表明PPAP2B基因变异与单向 协同流动促进内皮细胞PPAP2B。总体目标是阐明分子聚合性 内皮细胞miR92a-PPAP2B途径中的CAD遗传易感性和机械转导机制 这种方式的失调被认为是导致动脉粥样硬化的原因。我们建议的研究- 着装三个至关重要的问题：1)内皮细胞miR92a-PPAP2B途径是否存在因果关系 在体内调节动脉粥样硬化？我们用新的鼠标模型和创新的纳米颗粒在Aim 1中测试了这一点 选择性调控内皮细胞miR92a-PPAP2B信号转导。2)冠心病保护性等位基因在 Rs17114036影响PPAP2B的表达？在目标2中，我们从功能上描述了rs17114036-depedent PPAP2B中的增强子采用体内和体外互补的遗传方法。3)什么是Addi- KLF2在调节PPAP2B等位基因特异性表达中的作用？我们解决了对此进行动态监管的问题 Rs17114036-通过目标3中的KLF2进行机械和药物刺激的含有增强剂。

Project Summary Atherosclerotic arterial disease remains the leading cause of morbidity and mortality among Americans. Atherosclerosis preferentially develops in arterial sites such as curvatures and bifurcations, where endothelial cells are activated by local disturbed flow. Recent studies have suggested new genes and previously unsus- pected biology that mechanistically contribute to atherosclerosis; however, the interplay between genetic pre- disposition of atherosclerosis and flow-mediated endothelial functions remains to be elucidated. Genome-wide association studies (GWAS) and subsequent investigations identified PhosPhatidic-Acid- Phosphatase-type-2B (PPAP2B, also known as LPP3 or PLPP3) as a gene at chr 1p32.2 that significantly influences risk of coronary artery disease (CAD). Moreover, reduced endothelial PPAP2B is associated with increased CAD susceptibility. We recently discovered a new mechanism that links disturbed flow to reduction in endothelial PPAP2B through a microRNA-mediated mechano-transduction pathway: disturbed flow activates endothelial miR-92a, which in turn suppresses PPAP2B directly and also indirectly by inhibiting the transcription factor KLF2 that activates PPAP2B; reduced endothelial PPAP2B then leads to increased endothelial inflammation and compromised monolayer integrity. Furthermore, previous studies showed that systemic delivery of miR-92a inhibitor suppressed miR-92a in a wide range of tissues and lessened atherosclerosis in mice. Although these results suggest that flow-sensitive miR92a-PPAP2B pathway contributes to the athero-susceptible endothelial phenotype, the potentially causal relationship between endothelial miR92a-PPAP2B pathway and atherogenesis in vivo remains uncertain. Moreover, the mechanism by which the CAD-associated causal variant in PPAP2B exerts its effect and its potential roles in endothelial mechano-transduction remains unexplored. We have gathered very strong evidence that PPAP2B genetic variation interacts with unidirectional flow in concert to promote endothelial PPAP2B. The overall goal is to elucidate the molecular convergence of CAD genetic predisposition and mechano-transduction mechanisms in the endothelial miR92a-PPAP2B path- way, dysregulation of which is hypothesized to causatively promote atherosclerosis. Our proposed studies ad- dress three questions of fundamental importance: 1) Does the endothelial miR92a-PPAP2B pathway causally regulate atherosclerosis in vivo? We test this in Aim 1 with new mouse models and innovative nanoparticles to selectively modulate endothelial miR92a-PPAP2B signaling. 2) How does the CAD protective allele at rs17114036 influence PPAP2B expression? In Aim 2, we functionally characterize the rs17114036-depedent enhancer in PPAP2B employing complementary in vivo and in vitro genetic approaches. 3) What is the addi- tional role of KLF2 in regulating allele-specific PPAP2B expression? We address the dynamic regulation of this rs17114036-containing enhancer by mechanical and pharmacological stimuli via KLF2 in Aim 3.