Vascular Smooth Muscle Protein Quality Control and Aortic Aneurysm Formation

项目摘要 腹主动脉瘤（AAA）是一种毁灭性的疾病，由于高血压， 可能会造成致命的夹层和破裂目前还没有经过证实的药物治疗方法可以预防 AAA进展。VSMC变性在很大程度上导致了AAA的发病机制，但机制仍然存在 难以捉摸。泛素蛋白酶体系统（UPS）作为一种重要的蛋白质质量控制机制， 降解错误折叠的蛋白质和多余的正常蛋白质。UPS不足导致的蛋白毒性 功能已被广泛接受为多种退行性人类疾病的重要机制， UPS功能障碍在AAA中意义完全未知。我们在人AAA组织中的通路分析 揭示了UPS功能与AAA的关联。在AAA小鼠模型中，泛素化的 蛋白，UPS性能受损的标志，先于VSMC变性和AAA形成， 随着疾病进展而加重。这表明，UPS性能不足可能是一种新的 AAA病因的潜在机制。MYOCD是VSMC收缩基因程序的主开关。如何 MYOCD是受调节的，在AAA中的功能未知。VSMC中的批量RNA-seq显示UPS-1的富集。 除了与VSMC收缩相关的途径外，MYOCD上调的基因程序中也存在相关途径。 MYOCD的强制表达改善了UPS的性能，同时抑制了培养的VSMC的变性。 AAA形成早期的VSMC和VSMC特异性MYOCD转基因（Tg）小鼠。MYOCD 增加了UPS功能的两个关键参与者NFE 2L 1和KLHL 3的表达。这些证据 提示MYOCD在UPS功能中新作用。磷酸二酯酶（PDE），通过催化 cAMP和cGMP特异性地调节环核苷酸信号，在VSMC病理生理学中起关键作用 并且被证明是多种人类疾病的药物靶标。我们发现PDE 10A是最易诱导的PDE 在AAA形成过程中，MYOCD家族成员的表达受到抑制。PDE 10A的抑制 增加MYOCD蛋白，改善UPS性能，并抑制AAA形成。这些令人兴奋 初步研究结果支持了一个新的假设，即PDE 10A下调MYOCD蛋白会损害UPS 性能，导致VSMC退化和AAA。我们提出了三个目标来检验这一假设。要求1 将使用新的VSMC特异性Myocd敲除和Tg小鼠来确定MYOCD在AngII诱导的血管紧张素Ⅱ（Ang Ⅱ）表达中的功能。 AAA型号。目的2将使用一种新的UPS报告小鼠系和生化测定来确定 MYOCD调节的AAA中的UPS功能以及MYOCD如何调节UPS功能。目标3将决定如何 PDE 10促进MYOCD蛋白降解，PDE 10A在UPS性能和AAA形成中起作用。 该提案将首次阐述UPS性能在VSMC退化和AAA中的重要性 形成，并阐明了一种新的调控级联，包括可药物化的上游调节剂（PDE 10A）和 保护UPS性能和蛋白质稳态的下游效应子（MYOCD）。

PROJECT SUMMARY Abdominal aortic aneurysm (AAA) is a devastating disease carrying high morbidity and mortality due to the high likelihood of fatal dissection and rupture. There are currently no proven pharmaceutical treatments to prevent AAA progression. VSMC degeneration contributes largely to AAA pathogenesis, but the mechanism remains elusive. The ubiquitin proteasome system (UPS) serves as an essential protein quality control mechanism by degrading misfolded proteins and surplus normal proteins. While proteotoxicity resulting from insufficient UPS function has been widely accepted as an important mechanism for multiple degenerative human conditions, the implication of UPS dysfunction in AAA is completely unknown. Our pathway analysis in human AAA tissues revealed an association of UPS function with AAA. In a mouse model of AAA, accumulation of ubiquitinated proteins, a hallmark of impaired UPS performance, precedes VSMC degeneration and AAA formation and is exacerbated with disease progression. This suggests that inadequate UPS performance may act as a novel mechanism underlying AAA etiology. MYOCD is a master switch of VSMC contractile gene program. How MYOCD is regulated and functions in AAA is unknown. Bulk RNA-seq in VSMCs showed an enrichment of UPS- related pathways in the top MYOCD-upregulated gene programs besides those relevant to VSMC contraction. Forced expression of MYOCD improved UPS performance while suppressing VSMC degeneration in cultured VSMCs and VSMC-specific MYOCD transgenic (Tg) mice at the early stage of AAA formation. MYOCD increased the expression of NFE2L1 and KLHL3, two key players of UPS function. These lines of evidence suggest a novel role of MYOCD in UPS function. Phosphodiesterases (PDEs), by catalyzing the hydrolysis of cAMP and cGMP to specifically modulate cyclic nucleotide signaling, play critical roles in VSMC pathophysiology and are proven drug targets for multiple human diseases. We found that PDE10A was the most induced PDE family member during AAA formation, while MYOCD protein expression was suppressed. Inhibition of PDE10A increased MYOCD protein, improved UPS performance, and suppressed AAA formation. These exciting preliminary findings support a novel hypothesis that downregulation of MYOCD protein by PDE10A impairs UPS performance, leading to VSMC degeneration and AAA. We propose three Aims to test this hypothesis. Aim 1 will use novel VSMC-specific Myocd knockout and Tg mice to determine the function of MYOCD in AngII-induced AAA model. Aim 2 will use a novel UPS reporter mouse line and biochemical assays to determine the importance of UPS function in MYOCD-regulated AAA and how MYOCD modulates UPS function. Aim 3 will determine how PDE10 promotes MYOCD protein degradation and PDE10A functions in UPS performance and AAA formation. This proposal will address for the first time the importance of UPS performance in VSMC degeneration and AAA formation, and elucidate a novel regulatory cascade comprising a druggable upstream modulator (PDE10A) and a downstream effector (MYOCD) in safeguarding UPS performance and proteostasis.