CRISPR/Cas9 介导的 A4GALT 抑制可挽救源自人诱导多能干细胞的法布里病血管病变模型中的内皮细胞功能障碍。
CRISPR/Cas9-mediated suppression of A4GALT rescues endothelial cell dysfunction in a fabry disease vasculopathy model derived from human induced pluripotent stem cells.
机构信息
Transplantation Research Center, College of Medicine, The Catholic University of Korea, Seoul, South Korea.
Division of Nephrology, Department of Internal Medicine, Seoul St. Mary's Hospital, The College of Medicine, The Catholic University of Korea, South Korea.
出版信息
Atherosclerosis. 2024 Oct;397:118549. doi: 10.1016/j.atherosclerosis.2024.118549. Epub 2024 Aug 2.
BACKGROUND AND AIMS
The objective of this study was to investigate the efficacy of CRISPR/Cas9-mediated A4GALT suppression in rescuing endothelial dysfunction in Fabry disease (FD) endothelial cells (FD-ECs) derived from human induced pluripotent stem cells (hiPSCs).
METHODS
We differentiated hiPSCs (WT (wild-type), WTC-11), GLA-mutant hiPSCs (GLA-KO, CMC-Fb-002), and CRISPR/Cas9-mediated A4GALT-KO hiPSCs (GLA/A4GALT-KO, Fb-002-A4GALT-KO) into ECs and compared FD phenotypes and endothelial dysfunction. We also analyzed the effect of A4GALT suppression on reactive oxygen species (ROS) formation and transcriptome profiles through RNA sequencing.
RESULTS
GLA-mutant hiPSC-ECs (GLA-KO and CMC-Fb-002) showed downregulated expression of EC markers and significantly reduced α-GalA expression with increased Gb-3 deposition and intra-lysosomal inclusion bodies. However, CRISPR/Cas9-mediated A4GALT suppression in GLA/A4GALT-KO and Fb-002-A4GALT-KO hiPSC-ECs increased expression levels of EC markers and rescued these FD phenotypes. GLA-mutant hiPSC-ECs failed to form tube-like structure in tube formation assays, showing significantly decreased migration of cells into the scratched wound area. In contrast, A4GALT suppression improved tube formation and cell migration capacity. Western blot analysis revealed that MAPK and AKT phosphorylation levels were downregulated while SOD and catalase were upregulated in GLA-KO hiPSC-ECs. However, suppression of A4GALT restored these protein alterations. RNA sequencing analysis demonstrated significant transcriptome changes in GLA-mutant EC, especially in angiogenesis, cell death, and cellular response to oxidative stress. However, these were effectively restored in GLA/A4GALT-KO hiPSC-ECs.
CONCLUSIONS
CRISPR/Cas9-mediated A4GALT suppression rescued FD phenotype and endothelial dysfunction in GLA-mutant hiPSC-ECs, presenting a potential therapeutic approach for FD-vasculopathy.
背景和目的
本研究旨在探讨 CRISPR/Cas9 介导的 A4GALT 抑制在挽救 Fabry 病（FD）内皮细胞（FD-ECs）内皮功能障碍中的作用，这些细胞来源于人诱导多能干细胞（hiPSC）。
方法
我们将 hiPSC（WT（野生型）、WTC-11）、GLA 突变型 hiPSC（GLA-KO、CMC-Fb-002）和 CRISPR/Cas9 介导的 A4GALT-KO hiPSC（GLA/A4GALT-KO、Fb-002-A4GALT-KO）分化为 ECs，并比较 FD 表型和内皮功能障碍。我们还通过 RNA 测序分析了 A4GALT 抑制对活性氧（ROS）形成和转录组谱的影响。
结果
GLA 突变型 hiPSC-ECs（GLA-KO 和 CMC-Fb-002）表现出 EC 标志物表达下调，α-GalA 表达显著降低，Gb-3 沉积增加，溶酶体内包涵体增多。然而，CRISPR/Cas9 介导的 A4GALT 在 GLA/A4GALT-KO 和 Fb-002-A4GALT-KO hiPSC-ECs 中的抑制增加了 EC 标志物的表达水平，并挽救了这些 FD 表型。GLA 突变型 hiPSC-ECs 在管形成实验中未能形成管状结构，细胞向划痕伤口区域的迁移明显减少。相比之下，A4GALT 抑制提高了管形成和细胞迁移能力。Western blot 分析显示，GLA-KO hiPSC-ECs 中 MAPK 和 AKT 磷酸化水平下调，而 SOD 和过氧化氢酶上调。然而，A4GALT 的抑制恢复了这些蛋白的改变。RNA 测序分析表明，GLA 突变型 EC 的转录组发生了显著变化，特别是在血管生成、细胞死亡和细胞对氧化应激的反应方面。然而，这些在 GLA/A4GALT-KO hiPSC-ECs 中得到了有效恢复。
结论
CRISPR/Cas9 介导的 A4GALT 抑制挽救了 GLA 突变型 hiPSC-ECs 的 FD 表型和内皮功能障碍，为 FD 血管病变提供了一种潜在的治疗方法。
Zotero 插件