Parallel Processing of Multiple Stimuli in the Vascular Endothelium

The endothelium is a complex network of cells that lines the entire vasculature and controls virtually all cardiovascular functions. Changes in the behaviour of endothelial cells underlies almost all cardiovascular disease. To regulate cardiovascular function, the endothelium integrates hundreds of signals that provide constant instructions. Signals arrive from as close as neighbouring endothelial cells and underlying smooth muscle cells to substances circulating from the most remote outpost of the body. These signals provide endless streams of information to the endothelium that must be integrated and decoded. How this is achieved is not understood. Here, we show the endothelium manages a multitude of extracellular signals by using spatially‐distinct endothelial cells that are primed to detect specific extracellular signals. In response to each extracellular signal, cells generate intracellular messages with unique characteristics. When multiple extracellular signals are present messages are communicated across cells and computations carried out to generate a consensus. These endothelial mechanisms may underlie how signals that utilise only one messenger (Ca 2+ ) can generate different stimulus‐specific responses across a vast number of cells. To determine how signals evoked by multiple agonists (ACh, ATP, ADP & histamine) were transduced in the endothelium, we studied Ca 2+ signals evoked by the extracellular activators in intact arteries. Male Sprague‐Dawley rats were euthanized by CO 2 overdose and second order mesenteric arteries were extracted, cut open and pinned flat. The exposed endothelium was loaded with a Ca 2+ indicator, Cal‐520/AM and imaged using fluorescence microscopy. Changes in intracellular Ca 2+ in thousands of endothelial cells were measured simultaneously and a custom analysis package written in Python was used to analyse the data. For each agonist, full concentration response curves were carried out to determine the EC 25 concentrations (the concentration at which 25% of the cells were activated). Interestingly, the EC 25 concentration increased 10‐fold between ACh (7.5 nM) & ADP (61.1 nM) and 100 fold between ACh & ATP (627.1 nM) and also for ACh & histamine (1.1 μM). The Ca 2+ signal to the EC 25 for each agonist began in spatially‐distinct clusters and propagated between cells as Ca 2+ waves. There was no significant overlap in the cells that first responded to each agonist or indeed in the entire population response (P<0.05). The activation of spatially‐distinct cells permits the endothelium to selectively process separate information and functions in parallel. A critical aspect of endothelial transduction of extracellular signals is how a single ion can control a host of different responses. Previous studies have shown that changes in the amplitude, frequency and kinetics of a Ca 2+ signal within a cell can lead to the generation of different responses. By studying thousands of endothelial cells our study reveals another novel feature of Ca 2+ signaling. Each agonist, when applied at low concentrations (EC 25 ) evoked unique Ca 2+ signals. This may provide insight into how the response that is generated to different agonists is communicated between cells to give an endothelium wide response. These results suggest that spatially‐distinct, stimulus‐specific cells unravel the constant streams of information to the endothelium held in the multitude of extracellular signals. Support or Funding Information This work was supported by the Wellcome Trust and British Heart Foundation This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .