Abstract：
OBJECTIVE: 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.
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.
摘要：
目的：本研究的目的是研究CRISPR/Cas9介导的A4GALT抑制在挽救源自人诱导多能干细胞（hiPSCs）的法布里病（FD）内皮细胞（FD-ECs）的内皮功能障碍中的功效。
方法：我们分化了hiPSC（WT（野生型），WTC-11），GLA-突变型hiPSCs(GLA-KO,CMC-Fb-002),和CRISPR/Cas9介导的A4GALT-KOhiPSC（GLA/A4GALT-KO，Fb-002-A4GALT-KO）进入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-EC（GLA-KO和CMC-Fb-002）显示EC标记表达下调，α-GalA表达显著降低，同时Gb-3沉积和溶酶体内包涵体增加。然而,GLA/A4GALT-KO和Fb-002-A4GALT-KOhiPSC-EC中CRISPR/Cas9介导的A4GALT抑制增加了EC标志物的表达水平并拯救了这些FD表型。GLA-突变型hiPSC-EC在管形成测定中未能形成管状结构，显示细胞向划伤区域的迁移显著减少。相比之下,A4GALT抑制改善了管形成和细胞迁移能力。Westernblot分析显示GLA-KOhiPSC-EC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质的改变。RNA测序分析表明GLA突变体EC的显著转录组变化，尤其是在血管生成中，细胞死亡,和细胞对氧化应激的反应。然而,这些在GLA/A4GALT-KOhiPSC-ECs中有效恢复。
结论：CRISPR/Cas9介导的A4GALT抑制挽救了GLA突变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。
方法：我们分化了hiPSC（WT（野生型），WTC-11），GLA-突变型hiPSCs(GLA-KO,CMC-Fb-002),和CRISPR/Cas9介导的A4GALT-KOhiPSC（GLA/A4GALT-KO，Fb-002-A4GALT-KO）进入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-EC（GLA-KO和CMC-Fb-002）显示EC标记表达下调，α-GalA表达显著降低，同时Gb-3沉积和溶酶体内包涵体增加。然而,GLA/A4GALT-KO和Fb-002-A4GALT-KOhiPSC-EC中CRISPR/Cas9介导的A4GALT抑制增加了EC标志物的表达水平并拯救了这些FD表型。GLA-突变型hiPSC-EC在管形成测定中未能形成管状结构，显示细胞向划伤区域的迁移显著减少。相比之下,A4GALT抑制改善了管形成和细胞迁移能力。Westernblot分析显示GLA-KOhiPSC-EC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质的改变。RNA测序分析表明GLA突变体EC的显著转录组变化，尤其是在血管生成中，细胞死亡,和细胞对氧化应激的反应。然而,这些在GLA/A4GALT-KOhiPSC-ECs中有效恢复。
结论：CRISPR/Cas9介导的A4GALT抑制挽救了GLA突变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。
