A novel treatment of aortic disease in Marfan Syndrome targeting oxidative stress and PKG dysregulation

摘要 主动脉瘤和夹层是马凡综合征(MFS)最严重和最致命的表现， 而目前预防主动脉扩张的治疗方法只能起到一定的效果。MFS是由突变或 纤维蛋白-1(Fbn1)的缺失，Fbn1是包裹和连接弹性纤维的细胞外微纤维的一种成分 至主动脉中层的平滑肌细胞(SMC)。纤维蛋白功能降低改变了 细胞外基质和SMC，导致SMC凋亡和细胞外基质降解。此外， 信号改变会导致活性氧物种(ROS)和一氧化氮(NO)的产生增加， 增加氧化和亚硝化应激，并通过NO/cGMP途径激活蛋白激酶G(PKG)。 尽管ROS、活性氮种(RNS)和蛋白酪氨酸激酶(PKG)的激活与动脉瘤的形成有关 MFS的形成，既不是ROS/RNS的来源，也不是氧化/亚硝化应激对SMC的影响 功能是完全理解的。我们持有多项专利的维生素B12类似物可比酰胺是一种强效的 和多功能抗氧化剂，可以中和ROS和RNS，包括NO。在上一次授权期内，我们 显示具有激活的PKG1突变(Prkg1R177Q)的小鼠会导致胸主动脉瘤和夹层 在人类中，主动脉扩张与氧化应激增加和中层变性⸺弹性有关 纤维碎裂、基质金属蛋白酶活性升高、中层纤维化和SMC凋亡；可比胺 治疗完全阻止了这些变化。初步数据显示，可比胺也能降低主动脉 在MFS小鼠模型(Fbn1C1041G/)中扩张和防止弹性纤维断裂和SMC凋亡， 同时减少氧化应激的标志物和过量的PKG信号。我们假设ROS/RN增加 结合一氧化氮合酶(NOS2)升高引起的PKG激活，可导致SMC功能异常和主动脉 MFS的病理，优化的可比胺给药方案将防止主动脉扩张并改善 在患有MFS的小鼠中存活，特别是在与阻滞剂联合使用时。在目标1中，我们将确定机械- 人纤维蛋白1缺陷或突变(IPSC来源)中ROS/RNS过量产生的现象和后果 SMC，使用shRNA敲除和药理学方法抑制ROS/RNS生成酶和 PKG体外培养。我们将评估过量的一氧化氮合酶和蛋白激酶G活性在主动脉进展中的作用。 体内疾病，通过在Fbn1C1041G/小鼠中诱导SMC特异性的NOS2或PKG1基因敲除。我们还将测试 SMC特异性过氧化氢酶过表达对ROS的解毒是否能改善主动脉病理。在AIM2中，我们 将确定预防主动脉扩张和死亡的最佳可比胺剂量和开始时间 分别对中度(Fbn1C1041G/)和重度(Fbn1mgR/MGR)MFS小鼠进行解剖。此外，我们还将 将可比胺与β阻滞剂心得安联合使用，因为可比胺可防止主动脉中层退变， 而心得安可降低机械应力，但不影响中膜退行性改变。这些研究 可以显著改善MFS患者的主动脉疾病的治疗。

Summary Aortic aneurysms and dissections are the most serious and deadly manifestations of Marfan Syndrome (MFS), and current therapies to prevent aortic dilation are only moderately effective. MFS is caused by mutations or deletions in fibrillin-1 (Fbn1), a component of extracellular microfibrils, which surround and connect elastic fibers to smooth muscle cells (SMCs) in the aortic media. Reduced fibrillin function alters signaling between extracellular matrix and SMCs, resulting in SMC apopotosis and extracellular matrix degradation. In addition, the altered signaling leads to increased production of reactive oxygen species (ROS) and nitric oxide (NO), increasing oxidative and nitrosative stress and activating protein kinase G (PKG) via the NO/cGMP pathway. Although the increased ROS, reactive nitrogen species (RNS), and PKG activation contribute to aneurysm formation in MFS, neither the sources of ROS/RNS nor the effects of oxidative/nitrosative stress on SMC functions are fully understood. The vitamin B12 analog cobinamide, on which we hold several patents, is a strong and versatile antioxidant that can neutralize ROS and RNS, including NO. During the last grant period, we showed that mice with an activating PKG1 mutation (Prkg1R177Q) that causes thoracic aneurysms and dissections in humans, develop aortic dilation associated with increased oxidative stress and media degeneration⸺elastic fiber fragmentation, increased matrix metalloproteinase activity, media fibrosis, and SMC apoptosis; cobinamide treatment completely prevented these changes. Preliminary data show that cobinamide also reduces aortic dilation and prevents elastic fiber fragmentation and SMC apoptosis in a mouse model of MFS (Fbn1C1041G/+), while reducing markers of oxidative stress and excess PKG signaling. We hypothesize that increased ROS/RNS combined with PKG activation from increased NO synthase (NOS2) drive abnormal SMC functions and aortic pathology in MFS, and that an optimized dose schedule of cobinamide will prevent aortic dilation and improve survival in mice with MFS, especially when combined with a -blocker. In Aim 1, we will determine the mecha- nisms and consequences of excess ROS/RNS generation in human fibrillin1-deficient or mutant (iPSC-derived) SMCs, using shRNA knockdown and pharmacological approaches to inhibit ROS/RNS-generating enzymes and PKG in vitro. We will assess contributions of excess NO synthase and PKG activity to the progression of aortic disease in vivo, by inducing SMC-specific knockout of NOS2 or PKG1 in Fbn1C1041G/+ mice. We will also test whether ROS detoxification by SMC-specific catalase overexpression ameliorates aortic pathology. In Aim2, we will determine the optimal cobinamide dose and starting time to prevent aortic dilation and death from aortic dissections in mice with moderate (Fbn1C1041G/+) and severe (Fbn1mgR/mgR) MFS, respectively. In addition, we will combine cobinamide with the β-blocker propranolol, because cobinamide prevents aortic media degeneration, while propranolol reduces mechanical stress without affecting degenerative changes in the media. These studies could lead to considerably improved treatment of the aortic disease in patients with MFS.