Skin capillary endothelial cells form a network of spatiotemporally conserved Ca2+ activity

Swaminathan, A.; Gonzalez, D. G.; Matte-Martone, C.; Xu, F.; Simpson, D.; Monedero-Alonso, D.; Moore, J. L.; Mack, J. J.; Kam, C. Y.; Greco, V.

doi:10.1101/2025.08.15.669933

bioRxiv · 10.1101/2025.08.15.669933

Skin capillary endothelial cells form a network of spatiotemporally conserved Ca2+ activity

Swaminathan, A.·Gonzalez, D. G.·Matte-Martone, C.·Xu, F.·Simpson, D.·Monedero-Alonso, D.·Moore, J. L.·Mack, J. J.·Kam, C. Y.·Greco, V.

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Abstract

Ca2+ signaling and its regulation are important for endothelial cell (EC) function and signaling. Yet, the spatiotemporal organization of Ca2+ activity and its regulation across a vascular plexus is poorly understood in an in vivo mammalian context. To overcome this gap in knowledge, we developed an intravital imaging approach to resolve Ca2+ activity with single cell resolution in skin vasculature of adult mice via multiphoton microscopy. Here, we tracked thousands of Ca2+ events in the skin capillary plexus during homeostasis and observed signaling heterogeneity between ECs, with just over half displaying Ca2+ activity at any given time. Longitudinal tracking of the same mice revealed that the same capillary ECs maintain Ca2+ activity over days to weeks. Interestingly, activity dynamics, such as frequency and event duration, are not conserved at a single cell level but are maintained at an EC population level. Molecularly, conditional deletion of the gap junction protein Connexin 43 (Cx43cKO) in ECs lead to a subset of ECs displaying sustained Ca2+ activity, biasing signaling dynamics of the whole network towards chronically persistent activity over time. Sustained capillary Ca2+ activity resulted in vascular permeability and flow dysregulation. Lastly, through pharmacological targeting of known agonists/antagonists, we showed that inhibition of L-type Voltage Gated Ca2+ channels (VGCCs) non cell-autonomously restores Ca2+ activity, blood flow, and barrier function in Cx43cKO mice. Collectively, our work provides insight into the characteristics, extent, and regulation of Ca2+ activity in skin capillaries of live mice with unprecedented spatial and temporal resolution. Significance StatementCa2+ signaling in mammalian endothelial cells (ECs) locally regulates blood flow, force sensing, and vessel permeability. Past studies have investigated Ca2+ signaling during vascular remodeling and repair. However, there is a gap in our understanding of how tissue-level Ca2+ is spatiotemporally organized and regulated during homeostasis. Intravital imaging in skin vasculature of live mice reveals that a conserved network of ECs participates in tissue-wide Ca2+ signaling over weeks. How this network maintains itself over time requires cellular communication through gap junction protein Connexin 43 (Cx43). Loss of EC Cx43 leads to heightened plexus-wide Ca2+ activity, and vessel barrier and flow dysregulation. Inhibition of L-type Ca2+ channels non-cell autonomously restores the capillary Ca2+ landscape, and rescues both barrier and flow dysfunction.

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Swaminathan, A., Gonzalez, D. G., Matte-Martone, C., Xu, F., Simpson, D., Monedero-Alonso, D., Moore, J. L., Mack, J. J., Kam, C. Y., Greco, V.. 2025-08-20. Skin capillary endothelial cells form a network of spatiotemporally conserved Ca2+ activity. https://doi.org/10.1101/2025.08.15.669933

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