Investigation of the molecular mechanisms of vascular endothelial dysfunction in Hutchinson-Gilford Progeria Syndrome through in vitro 2D and 3D models

摘要 哈钦森-吉尔福德早衰症(HGPS)是一种罕见的遗传性疾病，具有以下特征 加速衰老。大多数HGPS病例是由LMNA中的从头突变引起的 导致孕激素的基因(C.1824C&GT；T；p.G608G)，这是一种有毒的层蛋白A蛋白变体。儿童 大多数人死于冠状动脉疾病或中风，平均年龄为14.6岁 好几年了。内皮功能障碍是心血管病理生物学的一个关键因素，因为 内皮通过激活eNOS维持血管内环境稳定和血管张力 用于生产一氧化氮(NO)。ENOS活性的紊乱会导致许多疾病，包括 动脉硬化。尽管在血管内皮细胞功能障碍的发病机制方面有丰富的知识 心血管疾病，对孕激素在内皮细胞破坏中的作用知之甚少 HGPS中的细胞功能。此外，有越来越多的证据表明，孕激素在 普遍衰老和心血管健康的许多方面。尤其是动脉粥样硬化 HGPS中的斑块与老年人身上发现的斑块相似。此外，血管僵硬在 HGPS很像正常衰老时看到的，在两个人群中都表现为 脉搏波速度。 利用人诱导多能干细胞来源的内皮细胞(IPSC-ECs)，我展示了 HGPS内皮型一氧化氮合酶表达水平和活性的降低 与他们的正常对照组进行比较。因此，一氧化氮生物利用度的枯竭 在静态和流体培养条件下的HGPS内皮细胞中。值得注意的是，IPSC派生的HGPS ECs 在形成微血管网络方面表现出eNOS依赖的功能缺陷，验证了 通过在健康的人脐静脉内皮细胞(HUVECs)中过表达孕激素。 我还发现了介导A×T到G×C转换的腺嘌呤碱基编辑程序(ABEmax)。 基因组DNA有效地纠正了HGPS突变，孕激素表达 显著降低到基础水平。此外，ABEmax挽救了核泡沫 HGPS IPSC-ECs的表型。因此，这项研究为HGPS提供了有价值的见解 与正常衰老相关的心血管病理和心血管疾病，并可能 引领HGPS治疗心血管疾病的新策略。

Abstract Hutchinson-Gilford progeria syndrome (HGPS) is a rare, genetic disorder with features of accelerated aging. The majority of HGPS cases caused by a de novo point mutation in the LMNA gene (c.1824C > T; p.G608G) that results in progerin, a toxic lamin A protein variant. Children with the disease mostly die from coronary artery diseases or strokes at an average age of 14.6 years. Endothelial dysfunction is a key contributor to the cardiovascular pathobiology as the endothelium maintains vascular homeostasis and vascular tone by activating eNOS responsible for nitric oxide (NO) production. Perturbation of eNOS activity causes many diseases including atherosclerosis. Despite the vast knowledge of endothelial dysfunction in the pathogenesis of cardiovascular disease, very little known about the role of progerin in the disruption of endothelial cell function in HGPS. Furthermore, there is accumulated evidence about the role of progerin in many aspects of generalized aging and cardiovascular health. Particularly, the atherosclerotic plaques in HGPS are similar to those found in aging individuals. Moreover, vascular stiffening in HGPS is much like that seen on normal aging that manifested in both populations by increased pulse wave velocity. Using human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs), I demonstrated the reduction of endothelial nitric oxide synthase (eNOS) expression level and activity in HGPS ECs compared to their normal controls. Consequently, the depletion of nitric oxide bioavailability in HGPS ECs both in static and fluidic culture conditions. Remarkably, iPSC-derived HGPS ECs exhibited eNOS dependent functional defects in forming microvascular networks that validated through over-expression of progerin in healthy human umbilical vein endothelial cells (HUVECs). I also found that Adenine Base Editor (ABEmax) that mediates the conversion of A×T to G×C in genomic DNA efficiently corrected the HGPS mutation, and the progerin expression was significantly reduced to the basal level. In addition, ABEmax rescued the nuclear blebbing phenotype of the HGPS iPSC-ECs. Thus, the study provides valuable insights into HGPS cardiovascular pathology and cardiovascular diseases associated with normal aging, and may lead to novel strategies to treat cardiovascular disease in HGPS.