TRPV4 protein, human

All the experiments were performed according to approved protocol by Northeast Ohio Medical University, IACUC. Mouse Lewis lung carcinoma (LLC) cells (2 × 10⁶) were subcutaneously injected in the flank region of wild type C57BL/6 mice (WT) or TRPV4 knockout mice in C57BL/6 background (KO). Tumor size was measured using calipers at 7, 14 and 21 days and tumor volume was calculated according to the formula V= 4/3*Pi*Length/2*(width/2)². At day 21, mice were euthanized and tumor tissues were collected and fixed for immunohistochemistry or stored at -80°C. To measure tumor angiogenesis, tumor tissue sections of 10 μm thicknesses were stained with anti-CD31 (PECAM-1) to visualize the microvessels, α-SMA to stain pericytes and DAPI to label the nuclei. Images were acquired using Olympus IX72 microscope and the microvessels density, diameter (Feret) and length were calculated using Image J software. For the in vivo drug experiments, six to eight mice/group were used and the animals were divided in to four groups: 1) WT (control) 2) WT + TRPV4 activator 3) WT + Cisplatin and 4) WT + TRPV4 activator + Cisplatin. Once the tumors were palpable (after 7 days), the mice were daily given an intraperitonial (i.p) injection of TRPV4 agonist GSK1016790A (10 μg/kg) to groups 2 and 4 until day 21. The anti-cancer drug Cisplatin (3 mg/kg/week) was administered i.p. once/week to groups 3, and 4, 3 days post treatment with TRPV4 activator, until day 21. The WT control received saline as a vehicle.

Ca²⁺ signals in ECs were imaged with a Revolution Andor confocal system (Andor Technology) composed of an upright Nikon microscope with a 60× water-dipping objective [numerical aperture (NA) 1.0] and an electron-multiplying CCD camera, as described previously (4 (link), 6 (link), 33 (link)). Briefly, images were acquired at 15 frames/s for arteries from GCaMP2 mice and 30 frames/s for Fluo-4–loaded arteries using Andor Revolution TL acquisition software (Andor Technology). Changes in emission associated with Ca²⁺ binding were detected by exciting at 488 nm with a solid-state laser and collecting emitted fluorescence using a 527.5/49-nm band-pass filter, with a center wavelength of 527.5 nm and a guaranteed minimum bandwidth of 49 nm. Ca²⁺ imaging experiments were performed at 36°C. TRPV4 Ca²⁺ sparklets were analyzed within a ROI defined by a 1.7-µm² box (5 × 5 pixels) positioned at a point corresponding to peak TRPV4 Ca²⁺ sparklet amplitude. In preparations from wild-type C57BL6 mice and AKAP150^−/−mice, ECs were loaded with Fluo-4 (10 µM) by incubating for 45 min at 30°C in the presence of pluronic acid (2.5 mg/ml) before imaging. The field of view was ~113 × 136 µm, corresponding to 14.5 ± 0.5 cells per field (n = 6 fields). CPA (30 µM, 15 min) was included to eliminate IP₃R-mediated Ca²⁺ signaling. The images were recorded before and 5 min after treatment with CCh, PMA, or OAG. Gö-6976 (1 µM, 10 min) was added before treatment with CCh, OAG, or PMA, and HC-067047 (1 µM, 10 min). Displayed F/F₀ traces were filtered using a Gaussian filter with a cutoff corner frequency of 3.98 Hz. Corresponding F/F₀ traces shown in figures represent the same ROIs before and after treatment. MEPs were identified as black holes in the inner elastic lamina, corresponding to an absence of autofluorescence (4 (link), 19 (link), 22 (link)). A diameter of 2 mm, the dimension of the smallest holes in autofluorescence images, was used as a low-end cutoff for defining a hole. Sparklet sites at MEPs were identified as those with peak fluorescence within 5 mm of the center of a hole in the inner elastic lamina. Changes in activity were expressed as fold change relative to controls.