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Lysosomal Ca 2+ signaling is emerging as a crucial regulator of endothelial Ca 2+ dynamics. Ca 2+ release from the acidic vesicles in response to extracellular stimulation is usually promoted via Two Pore Channels (TPCs) and is amplified by endoplasmic reticulum (ER)-embedded inositol-1,3,4-trisphosphate (InsP 3 ) receptors and ryanodine receptors. Emerging evidence suggests that sub-cellular Ca 2+ signals in vascular endothelial cells can also be generated by the Transient Receptor Potential Mucolipin 1 channel (TRPML1) channel, which controls vesicle trafficking, autophagy and gene expression. Herein, we adopted a multidisciplinary approach, including live cell imaging, pharmacological manipulation, and gene targeting, revealing that TRPML1 protein is expressed and triggers global Ca 2+ signals in the human brain microvascular endothelial cell line, hCMEC/D3. The direct stimulation of TRPML1 with both the synthetic agonist, ML-SA1, and the endogenous ligand phosphatidylinositol 3,5-bisphosphate (PI(3,5)P 2 ) induced a significant increase in [Ca 2+ ] i, that was reduced by pharmacological blockade and genetic silencing of TRPML1. In addition, TRPML1-mediated lysosomal Ca 2+ release was sustained both by lysosomal Ca 2+ release and ER Ca 2+ - release through inositol-1,4,5-trisphophate receptors and store-operated Ca 2+ entry. Notably, interfering with TRPML1-mediated lysosomal Ca 2+ mobilization led to a decrease in the free ER Ca 2+ concentration. Imaging of DAF-FM fluorescence revealed that TRPML1 stimulation could also induce a significant Ca 2+ -dependent increase in nitric oxide concentration. Finally, the pharmacological and genetic blockade of TRPML1 impaired ATP-induced intracellular Ca 2+ release and NO production. These findings, therefore, shed novel light on the mechanisms whereby the lysosomal Ca 2+ store can shape endothelial Ca 2+ signaling and Ca 2+ -dependent functions in vascular endothelial cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision, (Copyright © 2024 Brunetti, Berra-Romani, Conca, Soda, Biella, Gerbino, Moccia and Scarpellino.)

Bone formation involves a complex crosstalk between endothelial cells (EC) and osteodifferentiating stem cells. This functional interplay is greatly mediated by the paracrine and autocrine action of soluble factors released at the vasculature-bone interface. This study elucidates the molecular and functional responses triggered by this intimate interaction. In this study, we showed that human dermal microvascular endothelial cells (HMEC) induced the expression of pro-angiogenic factors in stem cells from human exfoliated deciduous teeth (SHED) and sustain their osteo-differentiation at the same time. In contrast, osteodifferentiating SHED increased EC recruitment and promoted the formation of complex vascular networks. Moreover, HMEC enhanced anaerobic glycolysis in proliferating SHED without compromising their ability to undergo the oxidative metabolic shift required for adequate osteo-differentiation. Taken together, these findings provide novel insights into the molecular mechanism underlying the synergistic cooperation between EC and stem cells during bone tissue renewal., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Petrillo, Genova, Chinigò, Roato, Scarpellino, Kopecka, Altruda, Tolosano, Riganti, Mussano and Munaron.)