Skin capillary endothelial cells form a network of spatiotemporally conserved Ca activity.

Unlabelled: Ca signaling and its regulation are important for endothelial cell (EC) functions, including local blood flow control, mechanotransduction, and barrier function. Yet the spatiotemporal organization of Ca activity and its regulation across a vascular plexus is poorly understood in an mammalian context, largely due to technical barriers. To overcome this gap in knowledge, we developed an approach to resolve Ca activity with single cell resolution in the skin vasculature of live adult mice by multi-photon imaging. Here, we tracked thousands of Ca events in the skin capillary plexus during homeostasis and observed signaling heterogeneity between ECs, with just over half displaying Ca activity over minutes. Longitudinal tracking of the same mice revealed that the same ECs maintain Ca activity over days to weeks. Interestingly, activity dynamics, such as frequency and event duration, are not conserved at a single cell level but at an EC population level. To identify the molecular underpinning of this spatiotemporal Ca activity, we conditionally deleted in ECs the most expressed gap junction protein - Connexin 43 (Cx43). We found that loss of Cx43 initially causes a subset of ECs to display sustained Ca activity and biases the dynamics of the whole network towards chronically persistent activity over time. Lastly, through pharmacological targeting of a small panel of known Ca mediators, we showed that inhibition of L-type Voltage Gated Ca channels largely restores physiological Ca activity after loss of Cx43, but has no effect on signaling dynamics in homeostatic settings.

Significance Statement: Ca signaling in mammalian endothelial cells (ECs) locally regulates blood flow, force sensing, and vessel permeability. In periods of vascular development and repair, there is a need for large-scale coordination in each of those functions. To understand what mechanisms drive collective Ca activity during vascular remodeling, we must first address the open question of how tissue-level Ca is spatiotemporally organized and regulated during homeostasis. Intravital imaging in skin vasculature of live mice reveals that a conserved set of cells orchestrates tissue-wide Ca from minutes to days to weeks. How this network maintains itself over time requires long-range communication through a gap junction protein, Connexin 43 (Cx43). Cx43 dysregulation subsequently recruits L-type Voltage Gated Ca channels to reshape the EC Ca landscape.