Abstract Body: The restoration of the microvasculature contributes to cardiovascular regeneration. Our previous work indicated that angiogenic transdifferentiation from fibroblasts to endothelial cells promotes vascular recovery from limb ischemia and a glycolytic shift is required for transdifferentiation. However, a comprehensive profile of the metabolic changes during transdifferentiation has not been performed, and the contribution of metabolic regulation in vascular recovery through transdifferentiation in vivo remains to be determined.
We used unbiased liquid chromatography-mass spectrometry (LC-MS) based metabolomics and identified uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) as the most upregulated metabolite during transdifferentiation during transdifferentiation. UDP-GlcNAc is the substrate for the post-translational modification, O-GlcNAcylation. O-GlcNActransferase (OGT) and O-GlcNAcase (OGA) are the pair of enzymes that add or remove this protein modification, respectively. We pharmacologically and genetically manipulated this pair of enzymes in vitro or in vivo and determined that inhibition of OGT impaired transdifferentiation, while inhibition of OGA enhanced transdifferentiation. Mechanistically, we provide evidence indicating that O-GlcNAcylation orchestrates epigenetic remodeling during transdifferentiation through modifying histone chaperone protein HIRA, which controls the de novo deposition of the non-canonical histone variant H3.3 to the chromatin, a critical event intricately linked to transcription activation. Furthermore, our findings are substantiated by lineage tracing and conditional knockout studies in mice, which underscore the pivotal role of O-GlcNAcylation in promoting angiogenic transdifferentiation, partly via activation of the HIRA-H3.3 pathway, and subsequent revascularization in vivo.
In conclusion, O-GlcNAcylation is a critical post-translational modification that enhances angiogenic transdifferentiation through a HIRA-dependent H3.3 deposition mechanism, thereby strengthening vascular recovery. This is the first study to demonstrate a clear regulatory role for O-GlcNAcylation in cellular reprogramming and uncover potential molecular pathways conducive to enhancing perfusion restoration in ischemic tissue. These findings offer a promising avenue for the development of novel therapeutic interventions targeting vascular ischemia.