Pressure induced changes in endothelial cell morphology and Ca2+ signaling in ex vivo pressurized resistance arteries revealed using 4D diSPIM myography and AI deep-learning

Abstract

The vascular endothelium is critical in promoting vasodilation of resistance-size arteries through Ca 2+ dependent pathways. Vascular endothelial cells (ECs) are exposed to mechanical stimulation through changing intravascular pressure and flow, yet it is unclear how these forces impact EC morphology and Ca 2+ signaling in pressurized arteries. Visualizing how the endothelium of an intact resistance-size vessel responds to changing physical forces requires isometric, sub-micron, resolution. Conventional confocal fluorescent microscope systems are limited by poor axial resolution. 4D dual inverted selective plane of illumination microscopy (diSPIM; 3i) uniquely provides isometric resolution at ~330 nm with high temporal resolution and minimal photobleaching or phototoxicity. We designed custom pressure myography chambers to allow simultaneous 4D diSPIM fluorescent imaging and vessel diameter tracking measurements via transmitted IR light (diSPIM Myography). We captured fluorescent images of ex vivo pressurized resistance-size mouse mesenteric arteries at 80, 60, 40, and 20 mm Hg before and after the generation of myogenic tone and in the absence of extracellular Ca 2+ . Representative images were annotated for ECs, nuclei, myoendothelial projections (MEPS), and Ca 2+ signals. Annotations were used as ground truth for CNN AI model generation and validation using Apeer (Zeiss). Arivis Vision4D (Zeiss) was used to run the AI model and quantify data. As static intravascular pressure is increased in arteries without tone (with or without Ca 2+ ), ECs increase in width as the vessel dilates. Myogenic tone minimizes pressure dilation and changes in EC width, however, ECs compress by up to 1 micron as pressure increases from 20 to 80 mm Hg. Additionally, increasing intravascular pressure in arteries with tone decreases local EC Ca 2+ signaling. The data demonstrate a relationship between intravascular pressure and EC shape and Ca 2+ signaling in intact resistance arteries under conditions of physiologic temperature, pressure, pH, and tone. diSPIM myography provides a method of studying the effects of physiologically relevant mechanical stimuli and the roles of various mechanosensitive mechanisms on vascular function. AI image annotation reduces analysis bias and increases the breadth of quantifiable microscopy data. DMC is supported by NIH R00HL133451 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.