Fabry disease is an X-linked lysosomal storage disorder caused by GLA mutations, leading to a deficiency in α-Galactosidase A activity and subsequent accumulation of globotriaosylceramide (Gb3). This accumulation contributes to progressive multiorgan dysfunction, with cardiovascular complications, particularly endothelial dysfunction and left ventricular hypertrophy being major drivers of disease morbidity and mortality. Although enzyme replacement therapy is currently the standard treatment, its effectiveness is limited in addressing advanced cardiovascular pathology. To better understand Fabry-associated vascular and cardiac phenotypes, an isogenic human induced pluripotent stem cell (hiPSC) model in which GLA was knocked out was developed using CRISPR/ Cas9. GLA-knockout (GLA-KO) hiPSCs were differentiated into endothelial cells (ECs) and cardiomyocytes (CMs) to evaluate disease-relevant phenotypes in vitro . GLA-KO ECs exhibited normal morphology and differentiation capacity but showed markedly impaired tube formation, high expression of inflammatory genes ICAM1, VCAM1, and SELE, and increased mitochondrial and cytoplasmic reactive oxygen species levels. GLA-KO CMs demonstrated enlarged cell size and nuclear translocation of NFATC4, consistent with hypertrophic remodeling. Together, these findings recapitulate key features of Fabry vasculopathy and cardiomyopathy in a genetically defined, human-derived system. This platform enables direct investigation of Gb3-induced oxidative and inflammatory mechanisms and provides a valuable model for the preclinical evaluation of therapeutic strategies targeting the cardiovascular manifestations of Fabry disease.

Introduction
Materials and Methods
    1. hiPSC culture and genome editing
    2. Endothelial differentiation and purification
    3. Cardiomyocyte differentiation
    4. Flow cytometry
    5. α-Galactosidase A enzymatic activity
    6. Immunocytochemistry and cell size measurement
    7. Quantitative reverse transcription polymerase chainreaction
    8. Reactive oxygen species measurement
    9. Statistical analysis
Results
    1. GLA-KO hiPSC-derived endothelial cells retaindifferentiation potential but lack GLA enzymaticactivity
    2. Hypertrophic phenotype in cardiomyocytes derivedfrom GLA-KO hiPSCs
    3. GLA-deficient endothelial cells exhibit impairedangiogenic function and elevated oxidative stress
Discussion
Funding
Conflicts of Interest
References

• Sang-Hyun Min(BK21, Infectious Disease Helathcare, Kyungpook National University, Daegu 41566, Republic of Korea, Department of Innovative Pharmaceutical Sciences, Kyungpook National University, Daegu 41566, Republic of Korea) Corresponding author
• Young-Kyu Kim(New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation (K-MEDI Hub), Daegu 41061, Republic of Korea)
• Sang-Wook Park(Department of Dental Bioscience, School of Dentistry, Chonnam National University, Gwangju 61186, Republic of Korea, Department of Oral Biochemistry, Dental Science Research Institute, School of Dentistry, Chonnam National University, Gwangju 61186, Republic of Korea) Corresponding author
• Yun Ju Choi(Department of Dental Bioscience, School of Dentistry, Chonnam National University, Gwangju 61186, Republic of Korea)