Fig 2: TRPV4 activation with GSK1016790A decreases trans‐endothelial electrical resistance (TER) in HUVEC and HPAEC monolayers. (a) Top: Time course of HUVEC monolayer impedance in control and after the addition of 30 nM GSK101, with or without HC06 (1 µM), and in the presence of HC06 alone. Bottom: Average TER values in control, after the addition of 30 nM GSK101 (with or without HC06 (1 µM)), and after the addition of HC06 alone. (b) Top: Time course of HPAEC monolayer impedance in control and after the addition of 30 nM GSK101, with or without HC06 (1 µM), and in the presence of HC06 alone. Bottom: Average TER values in control, after the addition of 30 nM GSK101 (with or without HC06 (1 µM)), and after the addition of HC06 alone. Data from three independent experiments are expressed as means ± SEM (***p < .001)

Fig 3: TRPV4 activation with GSK101 alters cytoskeleton (F‐Actin) and adherens junctions (VE‐Cadherin) in HUVECs and HPAECs. (a) Immunostainings of F‐Actin and VE‐Cadherin in HUVEC and HPAEC monolayers in control and 24 hr after the addition of 30 nM GSK101, with or without HC06 (1 µM), and in the presence of HC06 alone. (b) Top: Enlargement of GSK101‐induced effects on F‐Actin and VE‐Cadherin staining in HUVECs and HPAECs. Below: Plot profiles of F‐Actin or VE‐Cadherin fluorescence intensity corresponding to the yellow lines drawn on F‐Actin or VE‐Cadherin fluorescence images in control and after 24 hr stimulation with GSK101 (Mb: Membrane). (c) Graphs represent average membrane and intracellular F‐Actin or VE‐Cadherin fluorescence intensity in control, after 24 hr stimulation with GSK101 (with or without HC06 (1 µM)), and in the presence of HC06 alone. Data presented are the representative of three experiments (see material and methods) and expressed as means ± SEM (*** p < .001, ** p < .01, * p < .05)

Fig 4: Multiple pro‐inflammatory effects of Ca2+ entry through TRPV4 channels in venous and arterial endothelial cells. TRPV4 channel opening triggers cytoskeleton rearrangement/actin stress fiber formation, downregulation of adherent junction made of VE‐Cadherin and increased endothelial permeability, enhanced membrane expression of the adhesion molecule ICAM‐1, and apoptosis/necrosis. Therefore, Ca2+ influx through TRPV4 channels promotes the transition of both venous and arterial endothelial cells toward a pro‐inflammatory phenotype

Fig 5: Hypo-osmotic challenge inhibits IL-1β mediated NO release via a TRPV4 dependent pathway in isolated chondrocytes and cartilage explants. The TRPV4 antagonist, GSK205, suppresses the anti-inflammatory effects of hypo-osmotic challenge. Nitrite levels measured in the media for (A) isolated cells and (C) cartilage explants in hypo- or iso-osmotic media. Chondrocytes and cartilage explants were treated with and without IL-1β (1 ng/ml) for 24 h and 12 d respectively with and without the TRPV4 inhibitor GSK205 (10 μM). Hypo-osmotic challenge had no effect on cell viability as determined by (B) bright filed images of isolated chondrocytes (D) confocal microscopy of explants stained with Calcein-AM (live cells, green) and ethidium homodimer (dead cells, red). Scale bar represents 100 μm. Data represents mean ± SD for n = 6 separate wells (A) or n = 8 separate explants (C) using cells/explants isolated from 2 different donors. Statistics: Three-way ANOVA and post hoc Tukey's test. # represents statistically significant difference between IL-1β treated and corresponding untreated cells.