Indirect crosstalk between signalling pathways activated by CGRP and Piezo1 in human iPSC-derived endothelial cells relevant to migraine

Background It is becoming increasingly evident that the vasculature is implicated in migraine pathophysiology. Calcitonin gene-related peptide (CGRP) acts as one of the key migraine mediators through various mechanisms that includes endothelium-mediated cerebral vessel vasodilation. Endothelial cells express mechanosensitive Piezo1 channels and have been suggested to play a role in migraine pathophysiology. However, the crosstalk between these two migraine-related signalling pathways remains unclear. Methods We measured intracellular calcium (Ca 2+ ) in human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs), after exposure to Yoda1, a specific Piezo1 channel agonist, with and without CGRP. In addition, we investigated the effects of CGRP and Yoda1 on cellular remodelling by staining for focal adhesion (FA) protein paxillin using immunocytochemistry. Results Our data suggest that a one-hour sensitization of hiPSC-ECs with CGRP followed by application of Yoda1 leads to a higher intracellular Ca 2+ level compared to when Yoda1 and CGRP are acutely applied separately or combined, suggesting at least indirect crosstalk between the two signalling pathways in the vascular system. CGRP receptor antagonist BIBN4096 significantly reduced the intracellular Ca² + level under this sensitization protocol, confirming effective CGRP pathway blockade. The results also show that a one-hour sensitization of CGRP and Piezo1 activation affects cellular remodelling as evidenced by an increased number and area size of paxillin FA points per cell in hiPSC-ECs. Conclusions We have generated a human cell assay based on iPSC-derived endothelial cells and provided some evidence for crosstalk between mechanosensitive Piezo1 channels and CGRP in our hiPSC-EC system, which shows the potential for in vitro modelling of vascular implications relevant to migraine.