Abstract Body: Introduction: Induced pluripotent stem cells (iPSCs) derived endothelial cells (ECs) present a promising tool to advance tissue regeneration but require further optimization for implementation. Previously, we demonstrated that hyperoxia culture conditions (21% O₂) impaired iPSC-EC mitochondrial function and increased endothelial to mesenchymal transition (EndMT) compared to physoxia (4% O₂) (Carr, JVS, 2024). Here we investigate the role of metabolism, focusing on the multifaceted Neuropilin 1 (Nrp1) protein, in the regulation of iPSC-EC function.

Methods: Y6 human iPSCs were differentiated to ECs as previously described (Patsch Nat Cell Bio 2015). Resulting iPSC-ECs were cultured under varying oxygen conditions (hyperoxic 21% vs. physoxic 4%) for 2 weeks. Nrp1 was silenced with siRNA. Markers of cellular identity and metabolic profile were analyzed by Seahorse, immunofluorescence, and Western blot.

Results: Compared with physoxia in addition to increasing EndMT markers, hyperoxia significantly increased expression of Nrp1 (50% upregulation, p<0.05), PFKFB3, a key regulator of glycolysis (60% higher, p<0.05), and Poldip2 a mitochondrial protein associated with impaired EC function was elevated (25% P<0.05). Single-cell sequencing of iPSC-ECs under hyperoxic conditions broadly clustered into EndMT or activated ECs highly expressing MMPs; Nrp1 was broadly expressed in both clusters. Silencing Nrp1 shunted energy production away from mitochondrial respiration, while simultaneously reducing PFKFB3 expression by 40% (p<0.01). Immunofluorescent staining for Nrp1 revealed a high degree of colocalization with Glut1, a key EC glucose transporter. Co-immunoprecipitation pull down assay confirmed a direct interaction between Nrp1 and Glut1.

Conclusion: We observe a direct interaction between Nrp1 and Glut1, as well as a broader correlation between Nrp1 expression and metabolic shifts associated with EndMT in iPSC-ECs. Prior studies associated Nrp1 with angiogenic capacity in ECs. Our findings underscore the parallel shifts in metabolism to mobilize energy production that occur in both angiogenesis and EndMT. Further studies to better define the mechanism by which Nrp1 exerts this effect, and what parallel metabolic signals distinguish EndMT from angiogenesis, will be warranted.