Reversal of the Inflammatory Responses in Fabry Patient iPSC-Derived Cardiovascular Endothelial Cells by CRISPR/Cas9-Corrected Mutation

Abstract

The late-onset type of Fabry disease (FD) with GLA IVS4 + 919G > A mutation has been shown to lead to cardiovascular dysfunctions. In order to eliminate variations in other aspects of the genetic background, we established the isogenic control of induced pluripotent stem cells (iPSCs) for the identification of the pathogenetic factors for FD phenotypes through CRISPR/Cas9 genomic editing. We adopted droplet digital PCR (ddPCR) to efficiently capture mutational events, thus enabling isolation of the corrected FD from FD-iPSCs. Both of these exhibited the characteristics of pluripotency and phenotypic plasticity, and they can be differentiated into endothelial cells (ECs). We demonstrated the phenotypic abnormalities in FD iPSC-derived ECs (FD-ECs), including intracellular Gb3 accumulation, autophagic flux impairment, and reactive oxygen species (ROS) production, and these abnormalities were rescued in isogenic control iPSC-derived ECs (corrected FD-ECs). Microarray profiling revealed that corrected FD-derived endothelial cells reversed the enrichment of genes in the pro-inflammatory pathway and validated the downregulation of NF-κB and the MAPK signaling pathway. Our findings highlighted the critical role of ECs in FD-associated vascular dysfunctions by establishing a reliable isogenic control and providing information on potential cellular targets to reduce the morbidity and mortality of FD patients with vascular complications.

Fabry disease: Mechanism and therapeutics strategies

“…Recently, Song and co-workers reported that pro-inflammation pathway are activated within the cells with Fabry pathology. While the inflammatory responses can be reversed using gene therapy, further confirming the correlation between inflammatory activation and Fabry pathology (Song et al, 2021). Several molecular pathways have been reported to be attributed to the inflammatory activation of Fabry disease, including the nuclear factor kappa B (NF-κB) pathway, oxidative stress, and transforming growth factor-β (TGF-β) pathway.…”

Characterization of cellular phenotypes in neurons derived from induced pluripotent stem cells of male patients with Fabry disease

“…In addition, the mechanism of endothelial cell damage by lyso‐Gb3 includes cytokines, abnormalities in the immune system, and vascular damage caused by increased reactive oxygen species 19–21 . Furthermore, increases in inflammatory cytokines and ROS were observed in vascular endothelial cells differentiated from iPSCs derived from patients with FD 25,33 …”

Development of vascular disease models to explore disease causation and pathomechanisms of rare vascular diseases

“…This leads to an accumulation of globotriaosyceramide (GB3) in ECs of many tissues that can result in multiple organ failure, and dysfunction of endothelial cells in the microvasculature. Using this isogenic iPSC control line to derive ECs, they demonstrated a reversal of inflammatory responses, including decreased cytokine release and specific inflammatory markers such as ICAM, CCL2CCL5, CXCL1, IL6, IL8, monocyte inducible factor, and CXCL10 [ 35 ]. The importance of using isogenic controls is highlighted in this monogenetic rare vascular disease model, allowing the examination of pathologic phenotypes in a system without the modeling limitations of using healthy control lines, such as epigenetic, environmental, and genetic background differences.…”