BS38 Dna methylation proteins regulate endothelial colony-forming cell angiogenic dysfunction in hypoxia

Objective Cord blood-derived endothelial colony-forming cells (CB-ECFCs) show superior angiogenic capacity to mature endothelial cells, with low immunogenicity, making them the preferred cell-based therapy for cardiovascular diseases. However, their angiogenic potential is significantly reduced under ischaemic conditions. Recent research on endothelial cell-based therapeutics has shifted towards investigating the role of epigenetics in endothelial cell angiogenic function. Here, we aimed to understand effects of hypoxia on CB-ECFC angiogenic potential and associated signaling pathways with specific focus on DNA methylation. Methods: Human CB-ECFCs were isolated from healthy donors (n=15; 5 clones) and exposed to hypoxia (1% O2) or normoxia (21% O2) for 48h prior to transcriptome and protein expression analysis by RNA sequencing and Proteome Profiler array. Angiogenic potential was quantified by 3D Matrigel tubulogenesis assay, whilst levels of DNA methylation proteins were manipulated by siRNA knockdown and plasmid overexpression. Results: Experimental hypoxia markedly reduced angiogenic capacity of CB-ECFCs, indicated by reduced tube formation and downregulation of pro-angiogenic markers (e.g. eNOS, VEGFR2), together with repression of DNA methylation regulators, DNMT1 and UHRF1, versus normoxia controls. siRNA knockdown of DNMT1 or UHRF1 resulted in similarly reduced angiogenic capacity of CB-ECFCs, confirming a key role for these DNA methylation proteins. Detailed gene expression analysis revealed significant dysregulation of key angiogenesis and phosphokinase signaling pathways, linked to altered methylation protein levels. Further network analysis of differentially expressed genes highlighted novel upstream regulators of DNMT1 and UHRF1 as candidate targets to rescue reduced ECFC angiogenic capacity in hypoxia. Conclusion: Taken together, the results of this study highlight DNA methylation proteins as important ECFC regulators, which hold promise for selective targeting to protect angiogenic capacity and augment ECFC therapeutic potential for ischaemic cardiovascular disease. Conflict of Interest none