CRISPR/Cas9-mediated suppression of A4GALT rescues endothelial cell dysfunction in a fabry disease vasculopathy model derived from human induced pluripotent stem cells.

Item request has been placed! ×
Item request cannot be made. ×
loading   Processing Request
Read More Add to Saved list
  • Additional Information
    • Source:
      Publisher: Elsevier Country of Publication: Ireland NLM ID: 0242543 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1879-1484 (Electronic) Linking ISSN: 00219150 NLM ISO Abbreviation: Atherosclerosis Subsets: MEDLINE
    • Publication Information:
      Publication: Limerick : Elsevier
      Original Publication: Amsterdam, Elsevier.
    • Subject Terms:
      Induced Pluripotent Stem Cells*/metabolism ; Fabry Disease*/metabolism ; Fabry Disease*/genetics ; CRISPR-Cas Systems* ; Endothelial Cells*/metabolism ; alpha-Galactosidase*/genetics ; alpha-Galactosidase*/metabolism ; Galactosyltransferases*/genetics ; Galactosyltransferases*/metabolism ; Reactive Oxygen Species*/metabolism ; Cell Differentiation*; Humans ; Phenotype ; Mutation ; Trihexosylceramides/metabolism ; Cells, Cultured ; Transcriptome ; Signal Transduction ; Cell Line ; Oxidative Stress
    • Abstract:
      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.
      Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
      (Copyright © 2024 Elsevier B.V. All rights reserved.)
    • Contributed Indexing:
      Keywords: A4GALT; CRISPR/Cas9; Endothelial cells; Endothelial dysfunction; Fabry disease; hiPSCs
    • Accession Number:
      EC 3.2.1.22 (alpha-Galactosidase)
      EC 2.4.1.- (Galactosyltransferases)
      0 (Reactive Oxygen Species)
      EC 2.4.1.- (UDP-galactose-lactosylceramide alpha 1-4-galactosyltransferase)
      0 (Trihexosylceramides)
      71965-57-6 (globotriaosylceramide)
      EC 3.2.1.22 (GLA protein, human)
    • Publication Date:
      Date Created: 20240814 Date Completed: 20240920 Latest Revision: 20241009
    • Publication Date:
      20241010
    • Accession Number:
      10.1016/j.atherosclerosis.2024.118549
    • Accession Number:
      39141976