The Hidden Signaling Network of Blood Vessels

The cells lining your skin's capillaries are constantly sending each other messages—tiny pulses of calcium that help regulate blood flow, sense physical forces, and keep vessel walls intact. Scientists have known about this signaling for decades. What they didn't know, until now, is that it follows a remarkably organized pattern, one that persists across days and weeks, governed by a network of cells that have, in a sense, assigned themselves permanent roles.

A new study from Yale School of Medicine (YSM) and University of California, Los Angeles (UCLA), published June 23 in Proceedings of the National Academy of Sciences, reveals not only that this network exists, but what happens when it breaks down—and how it might be restored.

The study was done in the lab of Valentina Greco, PhD, Carolyn Walch Slayman Professor of Genetics at YSM and a Howard Hughes Medical Institute investigator, in close collaboration with the labs of Julia Mack, PhD, and Chen Yuan Kam, PhD, both at UCLA.

The team used genetically engineered mice whose endothelial cells—the cells that line blood vessels—glow green whenever calcium is active, allowing them to non-invasively watch the signaling in real time.

Across thousands of cells and multiple mice, the team found that, at any given time, roughly half the endothelial cells in a region were actively firing calcium signals; the other half were quiet.

“This observation piqued my interest,” says Mack. “We had previously reported that a similar proportion of endothelial cells cultured in a 2D monolayer under flow—about half—exhibited a similar calcium signaling activity. The finding pointed to differences within populations of cells that are present, where only some cells have an active calcium signaling state.”

When the researchers returned to the same mice 24 hours later, the same cells were active.

"We came back two weeks later, and those cells that were signaling on day 0 are still signaling, and the ones that were quiet then were still quiet," says Anush Swaminathan, an MD-PhD student at YSM and the paper’s first author. "So this seems to be conserved networks of cells over space and time that are signaling."

“We had previously identified that adult endothelial cells are remarkably stable with regards to their position within the vasculature,” says Kam. “Now, we can also appreciate that these positionally stable cells also retain their signaling identities. This provides insight as to how endothelial cells are able to integrate signals and communicate across interconnected blood vessels.”

Furthermore, individual cells varied considerably in how they signaled—the frequency and duration of calcium pulses shifted from day to day. Yet the overall statistical character of the network remained stable. The population, as a whole, maintained a consistent signature even as its members changed their behavior, almost like a cellular game of musical chairs among the signaling cells.

To understand how this stability is maintained, the team focused on Connexin 43 (Cx43), a protein that serves as a communication channel between neighboring cells. It is the most abundantly expressed connexin in skin endothelial cells, and the team hypothesized it might be coordinating the network's behavior.

When they genetically deleted Cx43 from endothelial cells, signaling didn't quiet down. It exploded.

"In the literature, what's been noted is when you get rid of these gap junction communication channels, essentially you're getting rid of the mouthpiece between these cells to talk to each other and signaling dies down," Swaminathan says. "In our case, we noticed that cells started screaming. There's more calcium activity across the entire tissue that lasts longer."

Some cells fired continuously for 17 minutes—a behavior never seen in typical mice. When the team tracked these mice over two weeks, the proportion of persistently firing cells grew. "Other cells that weren't initially behaving that way started joining in on the party," Swaminathan notes.

Further, blood flow in the capillaries increased and the vessels became leaky, allowing molecules to escape into surrounding tissue that should have stayed in the bloodstream. The endothelial barrier was failing.

The team screened a small panel of drugs targeting calcium channels, and one stood out: nifedipine, a medication used to treat high blood pressure. Applied topically to a mouse's paw, it restored normal calcium signaling, brought flow rates back to baseline, and repaired barrier function—all without touching the endothelial cells directly.

Nifedipine works on L-type voltage-gated calcium channels, and those channels aren't expressed in endothelial cells at all. They're expressed in pericytes—support cells that wrap around capillaries and possibly in smooth muscle cells upstream. The drug appears to calm those neighboring cells, which then send signals back to the endothelium, quieting the overactivity.

"When we use nifedipine to inhibit those channels in the tissue, then the surrounding cells actually have the opportunity to send signaling factors over to endothelial cells that then influence endothelial cell behavior," Swaminathan explains.

Swaminathan notes that preliminary experiments in younger, developing mice still showed a conserved signaling network, hinting that these cell identities may be set before the animals reach adulthood.

There are also open questions about what individual calcium events actually do in normal skin physiology. Unlike in the brain, where individual calcium pulses in capillaries correlate with changes in local blood flow, the team found no such correlation in the skin. It was only when the entire network became dysregulated that vascular function broke down.

"We still haven't answered the question: What are individual calcium events doing?" Swaminathan says. "Are they sensing things as they fly by? Are they responding to something?"

Those questions will drive the next phase of research. For now, the study establishes something that hasn't been shown before: that capillary endothelial cells form a spatially conserved, temporally stable signaling network in living mammals—and that network, when it goes awry, takes vascular function with it.