Calcium influx through TRPV4 channels modulates the adherens contacts between retinal microvascular endothelial cells

Abstract

The identity of microvascular endothelial (MVE) mechanosensors that sense blood flow in response to mechanical and chemical stimuli and regulate vascular permeability in the retina is unknown. Using immunohistochemistry, calcium imaging, electrophysiology, impedance measurements and vascular permeability assays, we show that the transient receptor potential isoform 4 (TRPV4) plays a major role in Ca /cation signalling, cytoskeletal remodelling and barrier function in retinal microvasculature in vitro and in vivo. Human retinal MVE cells (HrMVECs) predominantly expressed Trpv1 and Trpv4 transcripts, and TRPV4 was broadly localized to the plasma membrane of cultured cells and intact blood vessels in the inner retina. Treatment with the selective TRPV4 agonist GSK1016790A (GSK101) activated a nonselective cation current, robustly elevated [Ca ] and reversibly increased the permeability of MVEC monolayers. This was associated with disrupted organization of endothelial F-actin, downregulated expression of occludin and remodelling of adherens contacts consisting of vascular endothelial cadherin (VE-cadherin) and β-catenin. In vivo, GSK101 increased the permeability of retinal blood vessels in wild type but not in TRPV4 knockout mice. Agonist-evoked effects on barrier permeability and cytoskeletal reorganization were antagonized by the selective TRPV4 blocker HC 067047. Human choroidal endothelial cells expressed lower TRPV4 mRNA/protein levels and showed less pronounced agonist-evoked calcium signals compared to MVECs. These findings indicate a major role for TRPV4 in Ca homeostasis and barrier function in human retinal capillaries and suggest that TRPV4 may differentially contribute to the inner vs. outer blood-retinal barrier function.

Endothelial TRPV4 channels modulate vascular tone by Ca2+‐induced Ca2+ release at inositol 1,4,5‐trisphosphate receptors

“…These localized Ca 2+ signals were reported to activate endothelial Ca 2+ ‐activated K + channels (Sonkusare et al, ) and lead to endothelial hyperpolarization and vasodilation (Sonkusare et al, ). However, activation of TRPV4 channels is known to also control the orientation of endothelial cells, regulate endothelial permeability, and modulate the production of antithrombotic factors, each of which may require a more global [Ca 2+ ] increase throughout the cytoplasm (Noren et al, ; Phuong et al, ; Thodeti et al, ; Thoppil et al, ). In the present study, IP 3 R‐mediated Ca 2+ release generated propagating Ca 2+ waves and provided a mechanism by which TRPV4 channel activity, in the presence of a functioning Ca 2+ store, may generate large rises in Ca 2+ throughout the cytoplasm.…”

“…These localized Ca 2+ signals were reported to activate endothelial Ca 2+ -activated K + channels (Sonkusare et al, 2012) and lead to endothelial hyperpolarization and vasodilation (Sonkusare et al, 2012). However, activation of TRPV4 channels is known to also control the orientation of endothelial cells, regulate endothelial permeability, and modulate the production of antithrombotic factors, each of which may require a more global [Ca 2+ ] increase throughout the cytoplasm (Noren et al, 2016;Phuong et al, 2017;Thodeti et al, 2009;Thoppil et al, 2016).…”

TRPV4 Does Not Regulate the Distal Retinal Light Response

“…However, recent reports suggest that TRPV4 activation in Müller glia could play more significant roles under pathological circumstances associated with retinal detachment, photoreceptor degeneration, ischemia, and increased intraocular pressure (Ryskamp et al 2014; Taylor et al 2016). Moreover, TRPV4 is likely to play important functions in pressure, stretch, temperature, and volume sensing in the inner retina, including modulation of the blood-retina barrier permeability, responsiveness of RGC dendrites to intraocular pressure, and force-dependent remodeling of the optic nerve head in diseases such as diabetic retinopathy, ischemia, and glaucoma (Ryskamp et al 2011; Jo et al 2015; Phuong et al 2017; Krizaj 2016). …”

Polymodal Sensory Transduction in Mouse Corneal Epithelial Cells

“…The immunostaining protocols were as described previously. 33,34 Briefly, corneas were dissected along the sclero-corneal rim and fixed for 20 minutes in 4% ; mouse anti-Pannexin1, 1:100, Santa Cruz Biotech) were diluted (PBS with 2% BSA and 0.2% Triton X-100) and applied overnight at 4 0 C, followed by 1 hour incubation in fluorophoreconjugated goat anti-rabbit and mouse AlexaFluor 488 and/or 594 secondary antibodies (1:500, Life Technologies) at room temperature. The sections were mounted with DAPI-Fluoromount-G (Southern Biotech (Birmingham, AL, USA)).…”