Aberrant DNA methylation remodelling as a critical determinant of endothelial colony-forming cell angiogenic dysfunction in experimental hypoxia.

Objective: Cord blood-derived endothelial colony-forming cells (CB-ECFCs) are a valuable source for vascular repair with low immunogenicity provided their angiogenic potential can be effectively harnessed to combat harsh ischaemic conditions. Recent studies show that DNA methylation regulates endothelial cell angiogenic dysfunction under cardiovascular stress. We aimed to 1) investigate impact of DNA methylation changes on angiogenic capacity of CB-ECFCs under normoxia and hypoxia, and 2) target key impaired methylation targets to assess impact on post-ischaemic revascularisation in vivo, towards future development of innovative therapeutics. Methods: CB-ECFCs were exposed to hypoxic stress (1% O2) versus normoxia (21% O2) for 48h prior for assessment of angiogenic function by Matrigel. RNA and TWIST DNA methylation sequencing were conducted with integrated analysis to understand impact of DNA methylation remodelling on gene expression under hypoxia. siRNA knockdown/drug inhibition of key target genes was conducted to elucidate their specific effects on in vitro angiogenesis and in vivo post-ischaemic revascularisation. Results and conclusion: CB-ECFCs showed reduced angiogenic capacity under hypoxia, as reflected by Matrigel assay, and downregulation of angiogenic markers. Transcriptome data revealed that DNA repair pathways were significantly downregulated and linked to dysregulation of DNA methylation proteins, along with the upstream regulator, PARP1. DNMT1 and UHRF1 knockdown studies confirmed their role in regulating DNA repair proteins, whilst integrated analysis of methylome and transcriptome data led to novel discovery of SMARCA1, as an upstream regulator of DNA repair. Inhibition of PARP1, as a parallel upstream regulator identified from our IPA network, repressed DNA repair and methylation proteins whilst promoting angiogenic dysfunction in vitro and SMARCA1 activation, indicating PARP1-SMARCA1 linkage. PARP1 inhibition also reduced post ischaemic revascularization in vivo. In conclusion, this thesis provides novel insights into DNA methylation as a key epigenetic regulator of CB-ECFC angiogenesis whilst highlighting therapeutic potential for future treatment of ischaemic cardiovascular diseases. Thesis is embargoed until 31 July 2028.