Intermedin Enlarges the Vascular Lumen by Inducing the Quiescent Endothelial Cell Proliferation

Objective— Intermedin plays an important role in vascular remodeling and significantly improves blood perfusion, but the precise mechanism remains unclear. Herein, we aimed to define whether vascular lumen enlargement is responsible for the intermedin-increased blood perfusion and explore the underlying cellular and molecular mechanisms. Approach and Results— To study the role of intermedin, we generated the IMD-KO ( Adm2 −/− ) mice using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9) system. Intermedin significantly promoted vascular lumen enlargement in vitro (fibrin beads assay) and in vivo (murine retinas), which contributed to the improved blood perfusion in both physiological (retinal) and pathological (tumor) angiogenic models. We designed experiments to calculate the endothelial cell (EC) size and found that the lumen enlargement is because of EC proliferation but not because of a change in cell shape. ECs that construct vessel walls are considered quiescent cells because they are in a state of contact inhibition and show reduced responsiveness to VEGF (vascular endothelial growth factor). Using immunoprecipitation, Western blot assay, and fluorescent microscopy, we found that intermedin induced the formation of a signaling complex containing CRLR (calcitonin receptor-like receptor)/β-arr1 (β-arrestin1)/Src in ECs and promoted it internalizing into cytoplasm in a clathrin-dependent manner to activate downstream ERK1/2 (extracellular signal-regulated kinase 1/2). Importantly, this effect was not abrogated by cell–cell contacts of ECs. Through this mechanism, intermedin could reactivate the quiescent ECs to proliferate, resulting in continuous lumen expanding and a more effective blood perfusion. Conclusions— Our findings suggest a novel mechanism that may explain how quiescent ECs overcome the contact inhibition and regain the ability to proliferate for continuous vascular lumen enlargement.