4D diSPIM Myography Reveals Changes in Endothelial Cell Shape and Ca2+ Signaling in Response to Intravascular Pressure in ex vivo Resistance-sized Arteries

The vascular endothelium is critical in promoting vasodilation of resistance-sized arteries through Ca dependent pathways. The endothelium is exposed to mechanical stimulation through changing intravascular pressure and flow. Understanding how the endothelium integrates this information into a change in vascular function requires the ability to simultaneously measure changes vessel diameter and EC Ca signaling with high spatial and temporal resolution. We hypothesized that endothelial cell shape and Ca signaling is pressure dependent. To test this, we combined 4D Dual Inverted Selective Plane of Illumination Microscopy (diSPIM; 3i, CO) with pressure myography. This system allows for imaging of three-dimensional tissue preparations over time with very low photobleaching and phototoxicity, millisecond temporal resolution, and isometric 320 nm spatial resolution over a large imaging volume (330 x 330 x 150 mm). We designed and fabricated pressure myography chambers that position ex vivo pressurized arteries in the correct orientation for acquisition of high-resolution fluorescent data via diSPIM while using an inverted microscope base to continuously capture vessel diameter measurements via transmitted IR light. Resistance-sized mesenteric arteries from mice expressing an endothelium specific GECI are positioned between the diSPIM objectives and maintained at physiologic temperature and pH. We captured fluorescent images of whole pressurized arteries at 80, 60, 40, and 20 mmHg before and after the generation of myogenic tone and in the absence of extracellular Ca . As static intravascular pressure is increased, ECs increase in width and decrease in thickness. Myogenic tone minimizes changes in EC shape with changing pressure, however increasing static intravascular pressure decreases local EC Ca signaling. The data demonstrate a relationship between intravascular pressure and EC shape and Ca signaling in intact resistance arteries. This technique provides a method of studying the effects of physiologically relevant mechanical stimuli and the roles of various mechanosensitive mechanisms on vascular function.