The Hidden Signaling Network of Blood Vessels

What is it about?

Ca2+ signaling in mammalian endothelial cells (ECs) regulates blood flow, force-sensing, and vessel permeability. While past studies have investigated EC Ca2+ signaling in 2D monolayers and the 3D mouse brain, how Ca2+ signaling is organized over space and time across general vasculature remains poorly understood. Here, long-term intravital imaging of the same ECs across 3D skin capillaries of live mice reveals that a conserved EC network participates in Ca2+ signaling over time. How this network maintains itself requires communication through gap junction protein Connexin 43 (Cx43). Cx43 loss in ECs leads to increased Ca2+ activity, blood flow, and vascular permeability. Notably, chemical inhibition of L-type Ca2+ channels on cells within the tissue restores physiological Ca2+ activity, flow patterns, and barrier function.

Featured Image

Photo by Cristiano Pedroso-Roussado on Unsplash

Photo by Cristiano Pedroso-Roussado on Unsplash

Why is it important?

How Ca2+ activity is organized, maintained, and regulated across a vascular plexus in its native, unperturbed tissue environment is understudied. To our knowledge, our study represents the first time that Ca2+ activity of the same in vivo populations of mammalian ECs has been longitudinally tracked over minutes to days to weeks, demonstrating unexpected spatiotemporal patterns of endothelial Ca2+ activity and dynamics. Our uncovering of the spatiotemporal conservation of Ca2+ activity and its large-scale coordination emerges as critical features of skin capillaries. Heterogeneity of EC Ca2+ activity, which is conserved at a single-cell level over days to weeks, also suggests cellular heterogeneity in the same vascular compartment. This builds upon findings of EC heterogeneity in the same vascular compartment, across the vascular tree, and between organs, as a way for the endothelium to adapt to local environments. After loss of Cx43 in ECs, there is a loss of temporal maintenance of population-level behaviors that leads to increasing proportion of persistent signaling ECs over weeks, emphasizing a novel long-range, tissue-level regulatory role of Cx43 in temporal maintenance of plexus-wide EC Ca2+ signaling. The interplay we observed between L-type VGCC activity and Cx43 dysregulation also provides insight into how heterotypic cell interactions can reshape the Ca2+ landscape. Our discovery of sustained Ca2+ signaling after loss of Cx43 leading to flow and barrier dysfunction indicates that a set dynamic range of Ca2+ signaling across the network is necessary for homeostatic maintenance of vascular function, which when exceeded, leads to global flow dysregulation and barrier dysfunction. Ca2+ signaling in brain capillaries has been described to drive local blood flow changes. Our findings that homeostatic Ca2+ signaling is not correlated with apparent blood flow changes may indicate tissue-specific differences in Ca2+ regulation of flow and emphasize differences between central vasculature and regulation of general, peripheral vascular beds. Our work provides unprecedented spatial and temporal intravital imaging at single cell resolution to define the spatiotemporally conserved networks of capillary EC Ca2+ signaling and reveals that vascular function is compromised when this signaling is altered.