Intracellular Ca2+ variations were monitored on day 1 of vesseloids loaded with 5 μM (final concentration) Fluo-4 AM calcium probe (F23917, Thermo Fisher Scientific). Briefly, the dye solution was prepared as follows: 10 μg of powder was dissolved in 4 μl of Pluronic F-127 (P3000MP, Thermo Fisher Scientific), sonicated for 10 min, and diluted to the final concentration in standard medium. Vesseloids were incubated for 45 min at 37°C, then transferred to fresh medium, and imaged with a stereomicroscope (Nikon SMZ 18). We locally applied 0.1 μM ET-1 (E7764, Sigma-Aldrich) or 10 μM angiotensin II (A9525, Sigma Aldrich), and fluorescence was collected with a 10× objective and directed through a 525/50 band-pass filter to the camera. The acquired images were first processed for registration with available ImageJ plugin (StackReg). We then analyzed the registered images with a homemade LabView-based (National Instruments) software. The algorithm workflow was the following: (i) The background was subtracted using a region outside of the tube in each image. (ii) The temporal variation of fluorescence was then extracted pixel by pixel. (iii) Pixel fluorescence variations along the entire acquisition were used for automated segmentation of the cells (watershed based segmentation). (iv) For each segmented cell, time to peak and morphological features were computed.

The effect of ET-1 and angiotensin on in vivo contractility is classically shown by measuring the change in vascular tone (32). Since one advantage of the vesseloid model is that it is manipulable, contraction could be assessed here by handling vesseloids inside an imaging chamber and by directly measuring the diameter of the vesseloid before drug application and at maximal contraction. Image contrast was enhanced and normalized for the whole movie to improve the contrast at the border of the vesseloid and to compensate for photobleaching. A line with a width of 10 pixels was drawn perpendicularly to the main vesseloid axis and was used to measure the distance between the two walls. The percentage of contraction was defined as the relative diameter variation between resting and contracted states of the vesseloid.

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