Skip to main content
NCBI home page
British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2017 Dec 3;175(1):84–99. doi: 10.1111/bph.14072

Transient receptor potential vanilloid 4 channel regulates vascular endothelial permeability during colonic inflammation in dextran sulphate sodium‐induced murine colitis

Kenjiro Matsumoto 1,✉, Riho Yamaba 1, Ken Inoue 1, Daichi Utsumi 1, Takuya Tsukahara 1, Kikuko Amagase 1, Makoto Tominaga 2, Shinichi Kato 1
PMCID: PMC5740260  PMID: 29053877

Abstract

Background and Purpose

The transient receptor potential vanilloid 4 (TRPV4) channel is a non‐selective cation channel involved in physical sensing in various tissue types. The present study aimed to elucidate the function and expression of TRPV4 channels in colonic vascular endothelial cells during dextran sulphate sodium (DSS)‐induced colitis.

Experimental Approach

The role of TRPV4 channels in the progression of colonic inflammation was examined in a murine DSS‐induced colitis model using immunohistochemical analysis, Western blotting and Evans blue dye extrusion assay.

Key Results

DSS‐induced colitis was significantly attenuated in TRPV4‐deficient (TRPV4 KO) as compared to wild‐type mice. Repeated intrarectal administration of GSK1016790A, a TRPV4 agonist, exacerbated the severity of DSS‐induced colitis. Bone marrow transfer experiments demonstrated the important role of TRPV4 in non‐haematopoietic cells for DSS‐induced colitis. DSS treatment up‐regulated TRPV4 expression in the vascular endothelia of colonic mucosa and submucosa. DSS treatment increased vascular permeability, which was abolished in TRPV4 KO mice. This DSS‐induced increase in vascular permeability was further enhanced by i.v. administration of GSK1016790A, and this effect was abolished by the TRPV4 antagonist RN1734. TRPV4 was co‐localized with vascular endothelial (VE)‐cadherin, and VE‐cadherin expression was decreased by repeated i.v. administration of GSK1016790A during colitis. Furthermore, GSK106790A decreased VE‐cadherin expression in mouse aortic endothelial cells exposed to TNF‐α.

Conclusion and Implications

These findings indicate that an up‐regulation of TRPV4 channels in vascular endothelial cells contributes to the progression of colonic inflammation by increasing vascular permeability. Thus, TRPV4 is an attractive target for the treatment of inflammatory bowel diseases.

Abbreviations

DSS

dextran sulphate sodium

IBD

inflammatory bowel disease

LYVE1

lymphatic vessel endothelial hyaluronan receptor‐1

MAEC

mouse aortic endothelial cells

MPO

myeloperoxidase

TRP

transient receptor potential

TRPV

transient receptor potential vanilloid

VE

vascular endothelial

Introduction

The transient receptor potential vanilloid 4 (TRPV4) channel is a non‐selective cation channel that responds to mechanical, thermal and chemical stimuli, in addition to various endogenous ligands such as arachidonic acid metabolites (Watanabe et al., 2003). Among various transient receptor potential (TRP) channels, TRPV4 has gained increasing attention due to its widespread expression in mammalian tissues including the lung, liver, heart, kidney, CNS, skin, and salivary and sweat glands (Banner et al., 2011). Furthermore, TRPV4 is reportedly involved in the pathophysiology of inflammation, hypersensitivity and barrier dysfunction (Benemei et al., 2011).

The TRPV4 channel is also expressed in the gastrointestinal tract where it plays various physiological and pathophysiological roles (Vergnolle, 2014). Furthermore, neuronal TRPV4 contributes to visceral sensing and hypersensitivity in mice (Brierley et al., 2008; Mueller‐Tribbensee et al., 2015) and in patients with inflammatory bowel disease (IBD) (Brierley et al., 2008) and irritable bowel syndrome (Cenac et al., 2015). The expression of the TRPV4 channel has been demonstrated in nitric oxide neurons and it has been shown to be involved in regulating intestinal motility in the mouse enteric nervous system (Fichna et al., 2015). Epithelial TRPV4 channels can be detected in mice and IBD patients and were shown to contribute to the progression of inflammation (D'Aldebert et al., 2011; Yamawaki et al., 2014) and inhibition of motility (Fichna et al., 2015) in the large intestine. The activation of TRPV4 channels in epithelial cells is known to induce the release inflammatory cytokines and evoke intestinal inflammation in normal mice (D'Aldebert et al., 2011).

Several studies have demonstrated the expression of TRPV4 channels in the cardiovascular system including the endothelial cells of various organs (Randhawa and Jaggi, 2015). Additionally, endothelial TRPV4 regulates vascular permeability and/or relaxation in the lung (Sukumaran et al., 2013). Pharmacological blockade of TRPV4 was shown to result in a reduction in pro‐inflammatory cytokines and preserve the functions of the endothelium in a LPS‐induced murine model of sepsis (Dalsgaard et al., 2016). However, the localization and role of the TRPV4 channel in vascular endothelial (VE) cells in the gastrointestinal tract have not been elucidated.

In the present study we aimed to define the function and expression of the TRPV4 channel in colonic vascular endothelial cells during dextran sulphate sodium (DSS)‐induced colitis in TRPV4‐deficient mice using pharmacological tools, including a TRPV4 agonist and antagonist. The results demonstrated that TRPV4 expression in vascular endothelial cells contributes to the progression of colonic inflammation by increasing vascular permeability. Furthermore, we showed that the expression of endothelial TRPV4 increased markedly in the mucosa and submucosa of the colon, while that in the epithelia decreased during DSS‐induced colitis. The activation of TRPV4 decreased the expression of the major endothelial adhesion molecule VE‐cadherin in mouse aortic endothelial cells (MAEC) and the colon. These results suggest that endothelial TRPV4 is an attractive target for treating colitis.

Methods

Animals

Male C57BL/6 mice (8–10 weeks) weighing 22–27 g were purchased from Japan SLC Inc. (Shizuoka, Japan). TRPV4‐deficient mice (TRPV4 KO) were generated from a C57BL/6J background as described previously (Suzuki et al., 2003). All mice were maintained in plastic cages with free access to food and water and housed at 22 ± 1°C with a 12 h light/dark cycle. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015). The protocols were approved by the committee of the Ethics of Animal Research of Kyoto Pharmaceutical University (Permit numbers: 15‐12‐034 and 16‐12‐034). The number of animals used was kept to the minimum necessary for a meaningful interpretation of the data, and animal discomfort was kept to a minimum.

Group size

Equal group sizes were employed for all in vivo and in vitro experiments in this study. For studies using the in vivo model, the group size for each experimental condition was seven mice. For Western blot experiments of MAEC, five independent tests were used for each experimental condition.

Randomization

Wild‐type (WT) and/or TRPV4 KO mice were divided into groups according to the random comparison group methods in pharmacology. Vehicles and drugs were randomly assigned to each group prior to the start of the experiment.

Blinding and data normalization

Data were analysed by two observers who were blinded to the animal group assignment. Data files were labelled with the date and sample identifier. The data were analysed in this file format and subsequently assigned to the respective experimental condition using lab records. Values for vascular leakage were normalized to normal WT or normal vehicle‐treated mice, while those of protein expression determined by Western blotting were normalized to β‐actin protein expression.

Drugs and reagents

GSK1016790A, RN1734, Evans blue, N,N‐dimethylformamide and o‐dianisidine hydrochloride were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Captisol was purchased from Chemscene Ltd. (South Brunswick, NJ, USA). DMSO and TNF‐α were purchased from WAKO chemicals (Tokyo, Japan).

Induction of colitis

Colitis was induced in mice by oral administration of 2% DSS (molecular weight 36–50 kDa; MP Biomedicals, Irvine, CA, USA) in drinking water for 7 days. Normal mice (DSS‐untreated) only received normal drinking water. The stool score and histological damage were assessed by trained individuals blinded to the treatment groups as described previously (Theiss et al., 2009; Utsumi et al., 2016). Briefly, body weight loss, stool consistency and the presence of occult/gross blood were assessed daily. The stool characteristic was scored as follows: normal (score 0), soft with well‐formed pellets (score 1), very soft (score 2), diarrhoea (score 3) and severe diarrhoea (score 4). The occult blood was scored as follows: no blood (score 0), occult blood in the stool (score 1), traces of blood visible in the stool (score 2), gross bleeding (score 3) and rectal bleeding (score 4). These scores were added to obtain the stool score (maximum score of 8).

The distal colons from mice with DSS‐induced colitis were fixed in formalin and stained with haematoxylin and eosin. The histological scoring was performed based on three parameters as described by Theiss et al. (2009): the severity of inflammation, crypt damage and ulceration. The inflammation was scored as follows: rare inflammatory cells in the lamina propria (score 0), increased numbers of granulocytes in the lamina propria (score 1), confluence of inflammatory cells extending into the submucosa (score 2) and transmural extension of the inflammatory infiltrate (score 3). The crypt damage was scored as follows: intact crypts (score 0), loss of the basal one‐third (score 1), loss of the basal two‐thirds (score 2), entire crypt loss (score 3), a change in the epithelial surface with erosion (score 4) and confluent erosion (score 5). Ulceration was scored as follows: absence of ulcer (score 0), one or two foci of ulcerations (score 1), three or four foci of ulcerations (score 2) and confluent or extensive ulceration (score 3). These scores were added to obtain the histological score (maximum score of 11). A minimum of three sections of different parts of the colon were scored per animal.

Intracolonic administration of TRPV4 agonist

Animals were administered 2% DSS or normal drinking water for 7 days, and GSK1016790A (20 μg per mouse once daily) or vehicle were intracolonically administered as described below. Mice were anaesthetized with 2.5% or 1.5% isoflurane for the induction and maintenance of anaesthesia, respectively. The depth of anaesthesia was evaluated by pinching the animal's paw and all efforts were made to minimize suffering. Mice were treated with a single intracolonic injection of GSK1016790A (20 μg per mouse) in 50 μL PBS containing 20% DMSO (vehicle) or vehicle alone. The GSK1016790A dose was selected based on the intracolonic administration of another TRPV4 agonist, 4‐phorbol 12,13‐didecanoate (D'Aldebert et al., 2011). GSK1016790A is more potent and selective than 4‐phorbol 12,13‐didecanoate (Thorneloe et al., 2008).

Determination of myeloperoxidase (MPO) activity

The MPO activity was determined using o‐dianisidine hydrochloride, as previously described (Yasuda et al., 2011).

Bone marrow chimeras

Bone marrows were harvested from male TRPV4 KO or WT mice. Briefly, bone marrow cells were flushed out from the femur and tibia using 2–3 mL of PBS, filtered through a 70 μm nylon mesh, and counted for viability (>99% viability) before being injected into recipient mice. WT or TRPV4 KO recipient mice were irradiated with 9 Gy of X‐rays and injected i.v. with 5 × 106 bone marrow cells from WT or TRPV4 KO donor mice, yielding four experimental groups. On average, each donor mouse supplied a sufficient number of cells for two recipient mice.

After 6 weeks, recipient mice were treated using the acute DSS protocol described above. We confirmed bone marrow transfer by TRPV4 immunoreactivity in the bone marrow‐chimeric mice (Supporting Information Figure S1A). We observed TRPV4‐ and F4/80‐double labelled cells in the submucosal layer of WT and TRPV4 KO mice that had received bone marrow cells from WT mice (WT → WT or WT → KO), although double‐labelled cells were less abundant than F4/80‐single positive cells. In contrast, no TRPV4‐immunopositive cells were detected in the submucosal layer of WT and TRPV4 KO that had received bone marrow cells from TRPV4 KO mice (KO → WT or KO → KO). We also observed TRPV4‐ and F4/80‐double labelled cells in the bone marrow (Supporting Information Figure S1B).

Immunohistochemistry

On days 0 (normal), 4 and 7 following the initiation of DSS treatment, the animals were killed by CO2 gas inhalation. The colon was removed, washed with cold PBS and immersed in 4% paraformaldehyde for 2 h at 4°C. The tissues were cryoprotected overnight in 20% sucrose solution prior to being embedded in optimal cutting temperature compound (Sakura Finetek, Tokyo, Japan) mounting medium. They were then sectioned using a cryostat (Leica Instruments, Nussloch, Germany) at a thickness of 30 μm and thaw‐mounted onto Superfrost Plus slides (Matsunami, Osaka, Japan). Immunohistochemical procedures were performed as previously described by Matsumoto et al. (2011, 2012). Sources of all primary and secondary antibodies, as well as the optimized dilutions, are listed in Supporting Information Tables S1 and S2.

TRPV4 immunoreactivity was detected using the fluorescein conjugated tyramide amplification method (Perkin Elmer Life Sciences, Boston, MA, USA). We optimized the confocal microscopy condition according to the intensity of TRPV4 immunoreactivity on day 7 of DSS treatment. Using this condition, the intensity of epithelial TRPV4 immunoreactivity was much weaker than that observed in a previous study (D'Aldebert et al., 2011). Other molecules were detected by indirect staining with specific antibodies. The specificities of TRPV4 were demonstrated by the loss of immunostaining in TRPV4 KO mice (Figure 3B).

Microscopy and image analysis

Sections were viewed using a confocal microscope (A‐1R+; Nikon, Tokyo, Japan), and images were captured using Nikon NIS‐Elements AR 4.20.00 software. Multiple images in Z‐stacks were projected onto a single plane and reconstructed using the NIS‐Elements AR 4.20.00 software. For quantitative analyses, colon sections were viewed at 200× or 600× magnification using a confocal microscope, and quantitative determinations were made from three random locations for each mouse. For the analysis of TRPV4‐, CD31‐ and VE‐cadherin‐immunopositive area (%), active areas after interactive thresholding were measured using the NIS‐Elements AR 4.20.00 software image analysis system.

Western blot

Tissue preparation was performed as described by Eijkelkamp et al. (2007). Proteins were separated by 7.5% SDS‐PAGE and transferred onto PVDF membranes (Millipore, Bedford, MA, USA) by electroblotting. The membranes were stained with rabbit anti‐TRPV4 (1:300; Abcam, Cambridge, UK), rabbit anti‐VE‐cadherin (1:500; Abcam, Cambridge, UK), phosphorylated‐JNK2 antibody, JNK2 antibody (1:1000; Cell Signalling Technology, Inc., Danvers, MA, USA) and rabbit anti‐β‐actin (1:2000; Gene Tex, Gene‐Tex, San Antonio, TX, USA) antibodies. Immunoreactivity was detected by enhanced chemiluminescence (Perkin Elmer Life Sciences, Inc., Boston, MA, USA), and band density was determined using the VersaDoc5000/Dual (BioRad, Hercules, CA, USA).

Vascular leakage

Vascular leakage was determined using an Evans blue extravasation assay as described previously (Tan et al., 2010). Briefly, 0.2 mL of Evans blue dye (0.5% w.v‐1 in sterile PBS) was injected i.v. into anaesthetized mice and after 120 min, the animals were killed and extensively perfused with sterile PBS. There were no statistically significant differences in body weight among normal WT (24.7 ± 0.6 g), normal KO (24.2 ± 0.7 g), DSS WT (22.4 ± 0.6 g) and DSS KO (23.4 ± 0.6 g) mice in Figure 6A. Similarly, there were no statistically significant differences in body weight among normal + vehicle (24.4 ± 0.4 g), normal + GSK1016790A (23.7 ± 0.7 g), DSS + vehicle (21.7 ± 0.7 g), DSS + GSK1016790A (21.8 ± 0.6 g) and DSS + GSK1016790A + RN1734 (22.7 ± 0.5 g) in Figure 6C. Thus, the same volume of Evans blue dye (0.2 mL) was used for all mice. The colons were then harvested and weighed prior to dye extraction with N,N‐dimethylformamide (4 μL per 1 mg tissue weight) and absorbance reading at 620 nm. Data are expressed as fold increase in OD620 compared to normal mice. GSK1016790A (3 μg·kg−1) and RN1734 (100 μg·kg−1) were given as a single i.v. bolus. We used 1% DMSO / 20% Captisol (sulfobutyl ether‐β‐cyclodextrin) in saline as the vehicle, as described previously (Willette et al., 2008). The GSK1016790A dose selected should not affect the cardiovascular system, as demonstrated previously (Pankey et al., 2014; Gu et al., 2016).

Effect of TNF‐α or TRPV4 agonist in MAEC

MAEC were obtained by the BIOLOGICAL RESOURCE (Nishiyama et al., 2007) and maintained in Medium 199 (Life Technologies, Tokyo, Japan) containing 10% heat‐inactivated FBS, 100 U·mL−1 potassium penicillin G and 100 μg·mL−1 streptomycin sulphate. MAEC were incubated with 20 ng·mL−1 of TNF‐α or with 30 nM of GSK1016790A and 20 ng·mL−1 of TNF‐α for 0, 1, 3 or 6 h. Protein samples from MAEC were extracted using RIPA buffer supplemented with Complete Mini multi‐enzyme inhibitors and PhosSTOP phosphatase inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany).

Data and statistical analyses

The data and statistical analyses comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015). Data are presented as the mean ± SEM. Statistical analyses were performed with GraphPad Prism 6.07 (GraphPad Software, La Jolla, CA, USA) using one‐ or two‐way (genotype and treatment) ANOVA followed by Bonferroni's multiple comparison test for comparison of more than two groups, Student's t‐test for parametric data or Kruskal–Wallis one‐way ANOVA followed by Dunn's multiple comparison test for non‐parametric data. P values of <0.05 were regarded as statistically significant.

Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander et al., 2017a,b).

Results

The TRPV4 channel contributes to the exacerbation of intestinal inflammation in the murine colitis model

The addition of 2% DSS to drinking water produced diarrhoea and rectal bleeding (as indicated by the stool score) and body weight loss (Figure 1A, B). A shortening of the colon and a discernible increase in MPO activity and histological score were observed on day 7 of DSS treatment (Figure 1C–E). These responses were significantly attenuated in TRPV4 KO mice when compared to WT mice with DSS‐induced colitis.

Figure 1.

Figure 1

Open in a new tab

DSS‐induced colitis in WT and TRPV4‐deficient (KO) mice. Animals were untreated (normal) or exposed to 2% DSS for 7 days. Body weight (A) and stool score (B) were determined daily. Colon length (C), MPO activity (D) and histological score (E) were determined on day 7. Data are presented as the mean ± SEM, n = 7 mice per group. *P < 0.05 for comparisons with normal‐WT or normal‐KO mice. # P < 0.05 for comparisons with DSS‐WT mice. (F) Representative images of haematoxylin and eosin staining of WT and TRPV4 KO mice treated with DSS. Scale bars are 100 μm.

To confirm the role of TRPV4 in the pathogenesis of colonic inflammation, we also investigated the effects of a TRPV4 agonist GSK1016790A on DSS‐induced colitis (Figure 2). Repeated intracolonic administration of GSK1016790A for 7 days slightly decreased the body weight, increased the histological score in normal mice and exacerbated the severity of DSS‐induced colitis. A significant increase in body weight loss (Figure 2A), stool score (Figure 2B), shortening of the colon (Figure 2C), MPO activity (Figure 2D) and histological score (Figure 2E, F) was observed in mice that received GSK1016790A intracolonically daily when compared to mice that received vehicle alone. The vehicle‐treated mice showed a slight increase in body weight loss, stool score and histological score (Figure 2A, B, E).

Figure 2.

Figure 2

Open in a new tab

Effect of GSK1016790A (GSK), a TRPV4 agonist, on DSS‐induced colitis. Animals were untreated (normal) or exposed to 2% DSS or normal drinking water for 7 days. GSK (20 μg per mouse once daily) or vehicle was administered intracolonically. Body weight (A) and stool score (B) were determined daily. Colon length (C), MPO activity (D) and histological score (E) were assessed on day 7. Data are presented as the mean ± SEM, n = 7 mice per group. *P < 0.05 for comparisons with normal vehicle‐treated or normal GSK‐treated mice. # P < 0.05 for comparisons with DSS‐vehicle‐treated mice. (F) Representative images of haematoxylin and eosin staining of WT and TRPV4 KO mice treated with DSS. Scale bars are 100 μm.

We subsequently generated bone marrow chimeras to investigate the role of haematopoietic cells in the DSS colitis model (Figure 3). Bone marrow cells collected from WT or TRPV4 KO mice were transferred to irradiated WT or TRPV4 KO recipient mice, yielding four experimental groups. WT mice that received bone marrow cells from WT or TRPV4 KO mice (WT → WT or KO → WT) exhibited comparable body weight loss (Figure 3A), stool score (Figure 3B), shortening of the colon (Figure 3C), increase in MPO activity (Figure 3D) and histological score (Figure 3E, F). Similarly, KO mice that received bone marrow cells from WT or TRPV4 KO mice (WT → KO or KO → KO) showed comparable body weight loss (Figure 3A), stool score (Figure 3B), shortening of the colon (Figure 3C), increase in MPO activity (Figure 3D) and histological score (Figure 3E, F).

Figure 3.

Figure 3

Open in a new tab

DSS‐induced colitis in bone marrow‐chimeric mice. Bone marrow cells harvested from WT or TRPV4 KO mice were transferred to irradiated WT or TRPV4 KO recipient mice, yielding four experimental groups: (i) WT mice that received WT bone marrow cells (WT → WT); (ii) WT mice that received TRPV4 KO bone marrow cells (KO → WT); (iii) TRPV4 KO mice that received WT bone marrow cells (WT → KO); and (iv) TRPV4 KO mice that received TRPV4 KO bone marrow cells (KO → KO). Animals were untreated (normal) or exposed to 2% DSS for 7 days. Body weight (A) and stool score (B) were determined daily. Colon length (C), MPO activity (D) and histological score (E) were determined on day 7. Data are presented as the mean ± SEM, n = 7 mice per group. *P < 0.05 for comparisons with WT → WT mice. (F) Representative images of haematoxylin and eosin staining of the four experimental groups. Scale bars are 100 μm.

DSS treatment up‐regulates TRPV4 expression

The location of TRPV4 channels in normal and DSS‐treated mice on day 7 is shown on Figure 4. In normal mice, TRPV4 immunoreactivity was detected in epithelial cells of transverse and horizontal sections of the mouse colons (Figure 4A). However, on day 7 of DSS treatment an abundance of TRPV4 immunoreactivity was observed in the mucosal and submucosal layers of blood vessel‐like structures (Figure 4A). The TRPV4 immunoreactivity was abolished in TRPV4 KO mice (Figure 4B). Immunoreactivity in neurons was not clearly observed in either normal or DSS‐treated mice. Using Western blotting, we subsequently confirmed these immunohistochemistry results that showed TRPV4 channels are up‐regulated in the colon of mice after DSS treatment (Figure 4C).

Figure 4.

Figure 4

Open in a new tab

Alterations in TRPV4 expression in the colon of mice with DSS‐induced colitis. Animals were untreated (normal) or exposed to 2% DSS for 7 days (DSS), and the expression of TRPV4 was examined by immunohistochemistry (A, B) or Western blotting (C). (A) TRPV4 expression in the colon of normal and DSS mice as viewed in transverse (upper panel) and horizontal sections (lower panel). Scale bars are 50 μm (upper panel) and 20 μm (lower panel). Arrows indicate TRPV4 immunoreactivity. (B) TRPV4 expression in the mucosa of DSS‐treated WT and TRPV4 KO mice. (C) The expression of TRPV4 and β‐actin in the colon of normal and DSS mice. Data are presented as the mean ± SEM, n = 7 mice per group. A representative Western blot from one mouse is shown. *P < 0.05 for the comparison of normal with DSS mice.

TRPV4 immunoreactivity is up‐regulated in endothelial cells in DSS‐treated mice

Double‐labelling of TRPV4 with the epithelial marker keratin or endothelial cell marker CD31 were subsequently performed using transverse sections of the mouse colon on days 0 (normal), 4 and 7 of DSS treatment. Weak TRPV4 immunoreactivity co‐localizing with keratin was observed in epithelial cells on days 0 and 4 of DSS treatment (Figure 5A). However, DSS treatment up‐regulated the expression of TRPV4 that co‐localized with CD31 in the colon on days 4 and 7 of DSS treatment (Figure 5B). We also investigated the time‐dependent changes in TRPV4 and CD31 expression using quantitative immunohistochemical analysis on days 0, 4 and 7 of DSS treatment (Figure 5C). Immunoreactive areas in the mucosa, submucosal layer and muscle layer were quantified using computerized binary image analyses. A significant, 40‐fold, increase in TRPV4‐immunoreactive area was observed in the mucosa on day 7 of DSS treatment (10.7%) when compared to that observed in normal mice (0.276%). A significant, 40‐fold, increase in TRPV4‐immunoreactive area was also observed in the submucosal layer on days 4 (4.18%) and 7 (4.18%) when compared to that observed in normal mice (0.102%). However, no significant difference in TRPV4 area was observed in the muscle layer of normal and DSS‐treated mice at days 4 and 7. When compared to normal mice, DSS treatment significantly increased the CD31‐immunoreactive area, but the increase in immunoreactivity was less than twofold in the mucosa. There was no change in CD31‐immunoreactive area in the submucosal and muscle layers.

Figure 5.

Figure 5

Open in a new tab

Characterization of TRPV4 expression in the colon of normal mice or mice with DSS‐induced colitis. (A) Double labelling of TRPV4 (green) and keratin (red) in the colon of normal (DSS‐untreated), day 4 (DSS‐treated for 4 days) and day 7 (DSS‐treated for 7 days) mice. Arrowheads indicate the co‐localization of TRPV4 immunoreactivity with keratin. (B) Double labelling of TRPV4 (green) and CD31 (red) in the colon of normal (DSS‐untreated), day 4 (DSS‐treated for 4 days) and day 7 (DSS‐treated for 7 days) mice. Arrows indicate the co‐localization of TRPV4 immunoreactivity with CD31. (C) Quantitative analysis of TRPV4 and CD31 expressions in the mucosa, submucosa and muscle layers. Data are presented as the mean ± SEM, n = 7 mice per group. *P < 0.05 for comparisons with normal mice. (D) Double labelling of TRPV4 (green) and LYVE1, CD11b, Ly6B.2, CGRP or F4/80 (red) in the colon of mice treated with DSS for 7 days. Scale bars on panels (A) and (B) are 50 μm, and in (D) it is 20 μm.

Double‐labelling experiments of TRPV4 with the lymph node marker lymphatic vessel endothelial hyaluronan receptor‐1 (LYVE1), monocyte and macrophage marker CD11b, neutrophil marker Ly6B.2, sensory neuronal marker CGRP and murine macrophage marker F4/80 were also performed in DSS‐treated mice (Figure 5D). TRPV4 did not co‐localize with LYVE1 in vascular endothelial cells of the mucosal and submucosal layers of the colon. We found an abundant number of CD11b‐ and Ly6B.2‐positive cells in TRPV4‐immunopositive blood vessels. TRPV4 immunoreactivity did not co‐localize with CGRP in the DSS‐treated mice. TRPV4‐immunopositive cells were also detected in the submucosal layer and the staining co‐localized with that of F4/80.

TRPV4 activation increases vascular permeability in DSS‐treated mice

To investigate the relationship between TRPV4 and vascular permeability, we conducted vascular permeability assays using Evans blue dye (Figure 6). The DSS‐induced increase in vascular permeability in the colon of TRPV4 KO mice was significantly reduced when compared to that of WT mice (Figure 6A, B). We subsequently investigated the effect of the TRPV4 agonist GSK1016790A on vascular leakage in normal mice and those with DSS‐induced colitis (Figure 6C). The DSS‐induced increase in vascular permeability was further enhanced by a single i.v. injection of GSK1016790A, and this response was abolished by an i.v. injection of the TRPV4 antagonist RN1734.

Figure 6.

Figure 6

Open in a new tab

Changes in vascular leakage in the colon of mice with DSS‐induced colitis. (A) Vascular leakage in WT and TRPV4‐deficient (KO) mice under normal conditions or during DSS‐induced colitis. Data are presented as the mean ± SEM, n = 7 mice per group. *P < 0.05 for comparisons with the respective normal group. # P < 0.05 for the comparison with DSS‐treated WT mice. (B) Representative images of vascular leakage using Evans blue staining in WT and TRPV4 KO mice treated with DSS. (C) The effect of the TRPV4 agonist GSK1016790A (GSK) on the vascular leakage of normal mice and those with DSS‐induced colitis. GSK alone (3 μg·kg−1) or TRPV4 antagonist RN1734 (600 μg·kg−1) were given to mice i.v. Data are presented as the mean ± SEM, n = 7 mice per group. *P < 0.05 for comparisons with the respective normal group. # P < 0.05 for the comparison with DSS vehicle‐treated mice. + P < 0.05 for the comparison with DSS GSK‐treated mice.

DSS treatment decreases the expression of VE‐cadherin co‐localizing with TRPV4

We subsequently investigated changes in the expression of VE‐cadherin, the major endothelial adhesion molecule that regulates endothelial adherence junctions following the administration of the TRPV4 agonist GSK1016790A (GSK; Figure 7). We found that the structure of VE‐cadherin was compromised in mice treated with DSS‐vehicle or DSS‐GSK at day 7 (Figure 7A). TRPV4 co‐localized with VE‐cadherin in the mucosa of DSS‐vehicle and DSS‐GSK‐treated mice. However, the VE‐cadherin‐immunoreactive area was significantly smaller following repeated i.v. administration of GSK in colitis mice when compared to those treated with DSS‐vehicle (Figure 7B; top). At day 7, the TRPV4‐immunoreactive area in DSS‐vehicle and DSS‐GSK‐treated mice area was significantly larger than those of the normal groups (Figure 7B; bottom).

Figure 7.

Figure 7

Open in a new tab

Alterations in VE‐cadherin and TRPV4 channel expression in the colon of mice following i.v. administration of the TRPV4 agonist GSK1016790A (GSK). Animals were exposed to 2% DSS or normal drinking water for 7 days, while GSK (10 μg·kg−1 at days 2, 4 and 6) or vehicle (veh; at days 2, 4 and 6) was administered i.v. (A) The expression of TRPV4 and VE‐cadherin in the colon of normal vehicle‐treated, normal GSK‐treated, DSS vehicle‐treated and DSS GSK‐treated mice was assessed. Scale bars are 50 μm. Arrows indicate the co‐localization of TRPV4 immunoreactivity with VE‐cadherin. (B) Quantitative analysis of VE‐cadherin and TRPV4 expressions in the mucosa of the colon. Data are presented as the mean ± SEM, n = 7 mice per group. *P < 0.05 for comparisons with normal vehicle‐ or normal GSK‐treated mice. # P < 0.05 for the comparison with DSS vehicle‐treated mice.

TRPV4 activation decreases VE‐cadherin expression in MAEC

To confirm the alterations in TRPV4 expression in mouse endothelial cells during inflammation, we investigated changes in TRPV4 and VE‐cadherin expressions in MAEC following TNF‐α treatment Figure 8A, B, D, E). TRPV4 expression was significantly increased at 6 h post‐TNF‐α treatment compared to that at 0 h. However, there was no change in VE‐cadherin expression following TNF‐α treatment. We subsequently examined the effect of the TRPV4 agonist GSK1016790A in MAEC exposed to TNF‐α. At 6 h post‐TNF‐α/GSK1016790A treatment, the expression of TRPV4 was also increased, while that of VE‐cadherin was significantly decreased. We then investigated JNK phosphorylation in MAEC following TNF‐α, GSK1016790A or TNF‐α/GSK1016790A treatments (Figure 8C, F). The results showed that TNF‐α/GSK1016790A, but not TNF‐α or GSK1016790A alone, induced JNK phosphorylation.

Figure 8.

Figure 8

Open in a new tab

Alterations in TRPV4 and VE‐cadherin expressions in mouse aortic endothelial cells (MAEC). The effect of the TRPV4 agonist GSK1016790A (GSK) and TNF‐α in MAEC was evaluated. (A, B, D, E) The expression of TRPV4 and VE‐cadherin in MAEC following treatment with TNF‐α alone or with GSK. MAEC were incubated with 20 ng·mL−1 of TNF‐α or with 30 nM of GSK and 20 ng·mL−1 of TNF‐α for 0, 3 or 6 h. (C, F) JNK phosphorylation in MAEC following incubation with TNF‐α alone (20 ng·mL−1), GSK alone (30 nM) or TNF‐α with GSK for 0, 1 or 3 h. Data are presented as the mean ± SEM from five experiments. *P < 0.05 for comparisons with the respective 0 h group.

Discussion and conclusions

IBD, which includes ulcerative colitis and Crohn's disease, is an emerging health problem with its prevalence increasing worldwide (Strober et al., 2007). Endothelial cells play a key role in multiple aspects of chronic intestinal inflammation by regulating leukocytes that are migrating from the intravascular to interstitial space (Deban et al., 2008). The current study demonstrated for the first time that TRPV4 channels are highly up‐regulated in vascular endothelial cells of the colon during experimentally‐induced colitis. Vascular permeability was increased in a TRPV4‐dependent manner under this inflammatory condition. Our findings suggest that an alteration in TRPV4 expression in vascular endothelial cells contributes to the progression of colonic inflammation by increasing vascular permeability.

In previous studies, TRPV4 immunoreactivity was detected in epithelial cells of the intestines of mice and IBD patients (D'Aldebert et al., 2011; Yamawaki et al., 2014). Under inflammatory conditions, TRPV4 is up‐regulated in colonic epithelial cells (D'Aldebert et al., 2011), small intestinal epithelial cells (Yamawaki et al., 2014), pancreatic nerve fibres (Ceppa et al., 2010) and dorsal root ganglia (Vergnolle et al., 2010). In the present study, we detected TRPV4 immunoreactivity in epithelial cells in the colon of normal mice. However, the intensity of the immunoreactivity was relatively weak when compared to that observed in blood vessel endothelium in mice with DSS‐induced colitis. A significant, 40‐fold, increase in TRPV4 area was observed in the mucosa and submucosa at day 7 when compared to those of normal mice. Furthermore, TNF‐α treatment increased TRPV4 expression in MAEC in a time‐dependent manner. In a previous study it was shown that the pro‐inflammatory cytokine IL‐1β can also increase TRPV4 mRNA expression in mouse cerebral microvascular endothelial cells (Ma et al., 2008). These results suggest that the extent of endothelial TRPV4 expression is closely linked to the severity of intestinal inflammation.

The involvement of the TRPV4 channel in intestinal inflammatory processes has been demonstrated using selective TRPV4 agonist and/or antagonist in mice. D'Aldebert et al. (2011) reported that intracolonic administration of the TRPV4 agonist 4α‐phorbol 12,13‐didecanoate causes chronic inflammation in normal mice. Moreover, it was shown that systemic and intracolonic administration of the TRPV4 antagonist RN1734 alleviates 2,4,6‐trinitrobenzene sulfonic acid‐induced intestinal inflammation in mice (Fichna et al., 2012). Although these findings suggest that the TRPV4 channel is involved in inflammatory processes, studies assessing the effects of TRPV4 blockade using genetically deficient mice in an experimentally induced colitis are necessary to demonstrate the relationship between TRPV4 and intestinal inflammation. In the present study, we firstly demonstrated that TRPV4 KO mice were protected against DSS‐induced weight loss, leukocyte infiltration, colon shortening, diarrhoea, occult faecal blood and histological damage. We confirmed that repeated intracolonic administration of GSK1016790A exacerbated the severity of DSS‐induced colitis. To distinguish between the contribution of haematopoietic (bone marrow‐derived immune cells) and non‐haematopoietic cells, we performed bone marrow transplantation experiments in the DSS model. The results of the experiments suggest that TRPV4 channels in non‐haematopoietic cells (endothelial cells, sensory neuron and epithelial cells) play a dominant role in DSS‐induced colitis. Although the body weight loss and MPO activity increase were slightly attenuated in WT mice that received TRPV4 KO bone marrow grafts compared to those mice receiving bone marrow from WT mice, it is unlikely that TRPV4 channels in bone marrow‐derived immune cells contribute to DSS‐induced colitis.

As previously reported, neuronal TRPV4 plays an important role in gut functions, such as visceral sensing (Brierley et al., 2008; Mueller‐Tribbensee et al., 2015) and motility (Fichna et al., 2015). The functional expression of TRPV4 in the mouse enteric nervous system was previously reported (Fichna et al., 2015). Sensory neuropeptides (CGRP and substance P) released from TRPV4 neurons were shown to contribute to neurogenic inflammation in peripheral tissues (Vergnolle et al., 2010). The vasodilator effect of CGRP and substance P (Weidner et al., 2000) suggests these sensory neuropeptides are involved in the increase of vascular permeability. In the present study, TRPV4 immunoreactivity did not co‐localize with the sensory neuronal marker CGRP. D'Aldebert et al. (2011) also reported that capsaicin‐induced sensory denervation had no effect on the inflammatory responses in mouse colon induced by a TRPV4 agonist. We also investigated the role of TRPV1 using capsaicin‐desensitized mice under the same experimental condition. However, the inflammatory response induced by the 7‐day DSS treatment was exacerbated in capsaicin‐desensitized WT mice. Several studies have shown that DSS‐induced colitis is inhibited by capsaicin and resiniferatoxin desensitization (Kihara et al., 2003; Engel et al., 2012). However, we previously observed that capsaicin desensitization did not affect the severity of DSS‐induced colitis (Okayama et al., 2004). In another study it was shown that neonatal capsaicin denervation aggravates oxazolone‐induced colitis in mice (Lee et al., 2012). Thus far, we have not identified any differences between the results presented here and those obtained in capsaicin‐desensitized WT mice with DSS‐induced colitis. Further studies are needed to clarify the role of capsaicin desensitization.

Epithelial TRPV4 channels contribute to intestinal inflammation by regulating the epithelial barrier and inflammatory mediators in mice (D'Aldebert et al., 2011; Yamawaki et al., 2014). In agreement with a previous study (D'Aldebert et al., 2011), we found that the TRPV4 immunoreactive area co‐localized with an epithelial marker in normal mice. The activation of macrophage TRPV4 channels has been shown to contribute to ventilator‐induced lung injury (Hamanaka et al., 2010). Additionally, the TRPV4 channel was found to mediate neutrophil activation and acute lung injury (Yin et al., 2016). We found that TRPV4‐immunopositive cells co‐localized with the macrophage marker F4/80 (Supporting Information Figure S1A), which agrees with previous findings in alveolar macrophages (Gu et al., 2016). We also detected TRPV4‐ and F4/80‐double labelled cells in the bone marrow (Supporting Information Figure S1B). Scheraga et al. (2016) reported that TRPV4 protein is expressed in murine bone marrow‐derived macrophages. However, we hypothesized that most of the protective effects observed in mice with DSS‐induced colonic inflammation can be attributed to a genetic deficiency of TRPV4 in non‐haematopoietic cells, which was previously demonstrated in acute lung injury using bone marrow‐chimeric mice (Yin et al., 2016). Our results also showed that TRPV4‐ and F4/80‐double labelled cells were less abundant than F4/80‐single positive cells (Supporting Information Figure S1A). Therefore, we speculated that a lack of TRPV4 in macrophages has little effect in the acute colitis model. Nevertheless, further studies are needed to clarify the contributions of TRPV4 channels in epithelial and inflammatory cells to the progression of intestinal inflammation. The immunohistochemistry results and the assessments of the severity of intestinal inflammation suggest that the endothelial TRPV4 channel is involved in intestinal inflammation at a relatively severe stage.

The expression of various TRP channels such as TRP canonical 1, 4 and 6, TRPV4 and TRP melastatin 2 in endothelial cells has been shown to affect the vascular tone and permeability in various disorders (Yue et al., 2015). Among them, TRPV4 plays a key role in the function of the lung endothelial barrier (Morty and Kuebler, 2014). The TRPV4 agonist GSK1016790A was shown to increase vascular permeability in mouse lungs (Wandall‐Frostholm et al., 2015) and decrease transmembrane electrical resistance in human lung endothelial cells (Suresh et al., 2015). Dalsgaard et al. (2016) reported that pharmacological inhibition of TRPV4 prevents LPS‐induced endothelial dysfunction and increase in mesenteric artery permeability in mouse mesenteric arteries. Endothelial damage increases colonic vascular permeability, contributing to the pathogenesis of experimental colitis in rats and mice (Tolstanova et al., 2012). In this study, we found that the TRPV4 channel was up‐regulated in mouse endothelial cells during inflammatory conditions and a TRPV4 agonist increased vascular permeability in the colon of mice with DSS‐induced colitis, which was abolished by i.v. injection of a TRPV4 antagonist. In contrast, we did not observe endothelial TRPV4 immunoreactivity under physiological conditions, and the TRPV4 agonist did not affect vascular permeability in normal mice. These results suggest that the level of expression of TRPV4 channels is linked to the increase in vascular permeability following its activation. The TRPV4 channel can be activated by arachidonic acid and its subsequent metabolite 5,6‐epoxyeicosatrienoic acid (Watanabe et al., 2003; Vriens et al., 2005). Also 5,6‐epoxyeicosatrienoic acid has been demonstrated to increase vascular permeability through a mechanism involving endothelial the TRPV4 channel (Ivey et al., 1998). Therefore, we speculated that endothelial TRPV4 is activated by these intrinsic ligands under inflammatory conditions. In this study, we also observed TRPV4‐positive macrophages in mice with DSS‐induced colitis. Cytokines produced by inflammatory cells are widely known to increase vascular endothelial permeability (Sprague and Khalil, 2009). Further studies are needed to identify the relationship between TRPV4 channels in inflammatory and endothelial cells and their effect on vascular permeability under inflammatory conditions.

VE‐cadherin is the predominant component of endothelial adherence junctions that regulates intercellular gap formation. TNF‐α treatment was previously shown to reduce VE‐cadherin expression in human brain microvascular endothelial cells (Rochfort et al., 2016). Ca2+ signalling is critical for the disruption of VE‐cadherin junctions; therefore, it is also important in the mechanism of increased endothelial permeability (Tiruppathi et al., 2002). We found that TRPV4 activation by GSK1016790A treatment decreased VE‐cadherin expression in mice with DSS‐induced intestinal inflammation. Furthermore, TRPV4 activation by GSK106790A decreased VE‐cadherin expression in MAEC exposed to TNF‐α. These results suggest that TRPV4 activation leads to a decrease in VE‐cadherin expression under inflammatory conditions.

As previously reported, JNK signalling modulates VE‐cadherin expression and vascular endothelial permeability (Wu et al., 2008; Lopez‐Ramirez et al., 2012). Lastly, we investigated the mechanism behind the decrease in VE‐cadherin following TRPV4 activation under inflammatory conditions using MAEC. The results showed that TNF‐α/GSK1016790A, but not TNF‐α or GSK1016790A alone, induced JNK phosphorylation. Thus, it is unlikely that GSK1016790A alone can trigger the activation of the JNK signalling pathway. An up‐regulation of TRPV4 in response to TNF‐α may be required for the initiation of GSK1016790A‐induced JNK activation.

In conclusion, the up‐regulation of endothelial TRPV4 channels in colitis models suggests that these channels play a significant role in intestinal inflammation. TRPV4 activation decreased the major endothelial adhesion molecule VE‐cadherin in the mouse colon and MAEC. Our results suggest that TRPV4 antagonists, by affecting vascular permeability, may be beneficial for the treatment of chronic intestinal inflammation. Thus, the endothelial TRPV4 channel is an attractive target for the treatment of colitis.

Author contributions

K.M. and S.K. planned and designed the experiments. K.M., R.Y., K.I. and D.U. performed the experiments. K.M. and S.K. analysed the data. K.M. and S.K. wrote the manuscript. K.M., S.K., K.A. and M.T. reviewed and discussed the data.

Conflict of interest

The authors declare no conflicts of interest.

Declaration of transparency and scientific rigour

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.

Supporting information

Figure S1 TRPV4 expression in the colon of bone marrow‐chimeric mice and bone marrow cells. Bone marrow cells harvested from WT or TRPV4 KO mice were transferred to irradiated WT or TRPV4 KO recipient mice, yielding four experimental groups: 1) WT mice receiving WT bone marrow cells (WT → WT), 2) TRPV4 KO mice receiving WT bone marrow cells (WT → KO), 3) WT mice receiving TRPV4 KO bone marrow cells (KO → WT), and 4) TRPV4 KO mice receiving TRPV4 KO bone marrow cells (KO → KO). A) Double‐labelling of TRPV4 (green) and F4/80 (red) in the colon of mice treated with DSS for 7 days. Arrows indicate TRPV4 and F4/80 double‐positive cells. B) Double‐labelling of TRPV4 (green) and F4/80 (red) in bone marrow cells. Scale bars are 50 μm (A) and 10 μm (B).

Click here for additional data file. (9.1MB, tif)

Data S1 Supporting information.

Click here for additional data file. (44KB, doc)

Table S1 Primary antibodies.

Click here for additional data file. (44KB, doc)

Table S2 Secondary antibodies.

Click here for additional data file. (43.5KB, doc)

Acknowledgements

We thank Drs Makoto Suzuki and Atsuko Mizuno (Jichi Medical School) for providing the TRPV4‐deficient mice. We also thank the RIKEN BioResource Center for providing the MAEC. This work was supported in part by Grants‐in‐Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (#25860395 and 16K08287 to Kenjiro Matsumoto) and Takeda Science Foundation (to Kenjiro Matsumoto).

Matsumoto, K. , Yamaba, R. , Inoue, K. , Utsumi, D. , Tsukahara, T. , Amagase, K. , Tominaga, M. , and Kato, S. (2018) Transient receptor potential vanilloid 4 channel regulates vascular endothelial permeability during colonic inflammation in dextran sulphate sodium‐induced murine colitis. British Journal of Pharmacology, 175: 84–99. doi: 10.1111/bph.14072.

References

  1. Alexander SPH, Striessnig J, Kelly E, Marrion NV, Peters JA, Faccenda E et al (2017a). The Concise Guide To PHARMACOLOGY 2017/18: Voltage‐gated ion channels. Br J Pharmacol 174: S160–S194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alexander SPH, Fabbro D, Kelly E, Marrion NV, Peters JA, Faccenda E et al (2017b). The Concise Guide To PHARMACOLOGY 2017/18: Enzymes. Br J Pharmacol 174: S272–S359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Banner KH, Igney F, Poll C (2011). TRP channels: emerging targets for respiratory disease. Pharmacol Ther 130: 371–384. [DOI] [PubMed] [Google Scholar]
  4. Benemei S, Patacchini R, Trevisani M, Geppetti P (2011). TRP channels. Curr Opin Pharmacol 22: 18–23. [DOI] [PubMed] [Google Scholar]
  5. Brierley SM, Page AJ, Hughes PA, Adam B, Liebregts T, Cooper NJ et al (2008). Selective role for TRPV4 ion channels in visceral sensory pathways. Gastroenterology 134: 2059–2069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cenac N, Bautzova T, Le Faouder P, Veldhuis NA, Poole DP, Rolland C et al (2015). Quantification and potential functions of endogenous agonists of transient receptor potential channels in patients with irritable bowel syndrome. Gastroenterology 149: 433–444. [DOI] [PubMed] [Google Scholar]
  7. Ceppa E, Cattaruzza F, Lyo V, Amadesi S, Pelayo JC, Poole DP et al (2010). Transient receptor potential ion channels V4 and A1 contribute to pancreatitis pain in mice. Am J Physiol Gastrointest Liver Physiol 299: G556–G571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Curtis MJ, Bond RA, Spina D, Ahluwalia A, Alexander SPA, Giembycz MA et al (2015). Experimental design and analysis and their reporting: new guidance for publication in BJP. Br J Pharmacol 172: 3461–3471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. D'Aldebert E, Cenac N, Rousset P, Martin L, Rolland C, Chapman K et al (2011). Transient receptor potential vanilloid 4 activated inflammatory signals by intestinal epithelial cells and colitis in mice. Gastroenterology 140: 275–285. [DOI] [PubMed] [Google Scholar]
  10. Dalsgaard T, Sonkusare SK, Teuscher C, Poynter ME, Nelson MT (2016). Pharmacological inhibitors of TRPV4 channels reduce cytokine production, restore endothelial function and increase survival in septic mice. Sci Rep 6: 33841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Deban L, Correale C, Vetrano S, Malesci A, Danese S (2008). Multiple pathogenic roles of microvasculature in inflammatory bowel disease: a Jack of all trades. Am J Pathol 172: 1457–1466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Eijkelkamp N, Kavelaars A, Elsenbruch S, Schedlowski M, Holtmann G, Heijnen CJ (2007). Increased visceral sensitivity to capsaicin after DSS‐induced colitis in mice: spinal cord c‐Fos expression and behavior. Am J Physiol Gastrointest Liver Physiol 293: G749–G757. [DOI] [PubMed] [Google Scholar]
  13. Engel MA, Khalil M, Mueller‐Tribbensee SM, Becker C, Neuhuber WL, Neurath MF et al (2012). The proximodistal aggravation of colitis depends on substance P released from TRPV1‐expressing sensory neurons. J Gastroenterol 47: 256–265. [DOI] [PubMed] [Google Scholar]
  14. Fichna J, Mokrowiecka A, Cygankiewicz AI, Zakrzewski PK, Małecka‐Panas E, Janecka A et al (2012). Transient receptor potential vanilloid 4 blockade protects against experimental colitis in mice: a new strategy for inflammatory bowel diseases treatment? Neurogastroenterol Motil 24: e557–e560. [DOI] [PubMed] [Google Scholar]
  15. Fichna J, Poole DP, Veldhuis N, MacEachern SJ, Saur D, Zakrzewski PK et al (2015). Transient receptor potential vanilloid 4 inhibits mouse colonic motility by activating NO‐dependent enteric neurotransmission. J Mol Med (Berl) 93: 1297–1309. [DOI] [PubMed] [Google Scholar]
  16. Gu QD, Moss CR 2nd, Kettelhut KL, Gilbert CA, Hu H (2016). Activation of TRPV4 regulates respiration through indirect activation of bronchopulmonary sensory neurons. Front Physiol 7: 65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hamanaka K, Jian MY, Townsley MI, King JA, Liedtke W, Weber DS et al (2010). TRPV4 channels augment macrophage activation and ventilator‐induced lung injury. Am J Physiol Lung Cell Mol Physiol 299: L353–L362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ivey CL, Stephenson AH, Townsley MI (1998). Involvement of cytochrome P‐450 enzyme activity in the control of microvascular permeability in canine lung. Am J Physiol 275: L756–L763. [DOI] [PubMed] [Google Scholar]
  19. Kihara N, de la Fuente SG, Fujino K, Takahashi T, Pappas TN, Mantyh CR (2003). Vanilloid receptor‐1 containing primary sensory neurones mediate dextran sulphate sodium induced colitis in rats. Gut 52: 713–719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kilkenny C, Browne W, Cuthill IC, Emerson M, Altman DG, NC3Rs Reporting Guidelines Working Group (2010). Animal research: reporting in vivo experiments: the ARRIVE guidelines. Br J Pharmacol 160: 1577–1579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lee J, Yamamoto T, Kuramoto H, Kadowaki M (2012). TRPV1 expressing extrinsic primary sensory neurons play a protective role in mouse oxazolone‐induced colitis. Auton Neurosci 166: 72–76. [DOI] [PubMed] [Google Scholar]
  22. Lopez‐Ramirez MA, Fischer R, Torres‐Badillo CC, Davies HA, Logan K, Pfizenmaier K et al (2012). Role of caspases in cytokine‐induced barrier breakdown in human brain endothelial cells. J Immunol 189: 3130–3139. [DOI] [PubMed] [Google Scholar]
  23. Ma YY, Huo HR, Li CH, Zhao BS, Li LF, Sui F et al (2008). Effects of cinnamaldehyde on PGE2 release and TRPV4 expression in mouse cerebral microvascular endothelial cells induced by interleukin‐1beta. Biol Pharm Bull 31: 426–430. [DOI] [PubMed] [Google Scholar]
  24. Matsumoto K, Hosoya T, Tashima K, Namiki T, Murayama T, Horie S (2011). Distribution of transient receptor potential vanilloid 1 channel‐expressing nerve fibers in mouse rectal and colonic enteric nervous system: relationship to peptidergic and nitrinergic neurons. Neuroscience 172: 518–534. [DOI] [PubMed] [Google Scholar]
  25. Matsumoto K, Lo MW, Hosoya T, Tashima K, Takayama H, Murayama T et al (2012). Experimental colitis alters expression of 5‐HT receptors and transient receptor potential vanilloid 1 leading to visceral hypersensitivity in mice. Lab Invest 92: 769–782. [DOI] [PubMed] [Google Scholar]
  26. McGrath JC, Lilley E (2015). Implementing guidelines on reporting research using animals (ARRIVE etc.): new requirements for publication in BJP. Br J Pharmacol 172: 3189–3193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Morty RE, Kuebler WM (2014). TRPV4: an exciting new target to promote alveolocapillary barrier function. Am J Physiol Lung Cell Mol Physiol 307: L817–L821. [DOI] [PubMed] [Google Scholar]
  28. Mueller‐Tribbensee SM, Karna M, Khalil M, Neurath MF, Reeh PW, Engel MA (2015). Differential contribution of TRPA1, TRPV4 and TRPM8 to colonic nociception in Mice. PLoS One 10: e0128242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nishiyama T, Mishima K, Ide F, Yamada K, Obara K, Sato A et al (2007). Functional analysis of an established mouse vascular endothelial cell line. J Vasc Res 44: 138–148. [DOI] [PubMed] [Google Scholar]
  30. Okayama M, Tsubouchi R, Kato S, Takeuchi K (2004). Protective effect of lafutidine, a novel histamine H2‐receptor antagonist, on dextran sulfate sodium‐induced colonic inflammation through capsaicin‐sensitive afferent neurons in rats. Dig Dis Sci 49: 1696–1704. [DOI] [PubMed] [Google Scholar]
  31. Pankey EA, Zsombok A, Lasker GF, Kadowitz PJ (2014). Analysis of responses to the TRPV4 agonist GSK1016790A in the pulmonary vascular bed of the intact‐chest rat. Am J Physiol Heart Circ Physiol 306: H33–H40. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Randhawa PK, Jaggi AS (2015). TRPV4 channels: physiological and pathological role in cardiovascular system. Basic Res Cardiol 110: 54. [DOI] [PubMed] [Google Scholar]
  33. Rochfort KD, Collins LE, McLoughlin A, Cummins PM (2016). Tumour necrosis factor‐α‐mediated disruption of cerebrovascular endothelial barrier integrity in vitro involves the production of proinflammatory interleukin‐6. J Neurochem 136: 564–572. [DOI] [PubMed] [Google Scholar]
  34. Scheraga RG, Abraham S, Niese KA, Southern BD, Grove LM, Hite RD et al (2016). TRPV4 mechanosensitive ion channel regulates lipopolysaccharide‐stimulated macrophage phagocytosis. J Immunol 196: 428–436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Southan C, Sharman JL, Benson HE, Faccenda E, Pawson AJ, Alexander SPH et al (2016). The IUPHAR/BPS guide to PHARMACOLOGY in 2016: towards curated quantitative interactions between 130 protein targets and 6000 ligands. Nucl Acids Res 44: D1054–D1068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sprague AH, Khalil RA (2009). Inflammatory cytokines in vascular dysfunction and vascular disease. Biochem Pharmacol 78: 539–552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Strober W, Fuss I, Mannon P (2007). The fundamental basis of inflammatory bowel disease. J Clin Invest 117: 514–521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sukumaran SV, Singh TU, Parida S, Narasimha Reddy CE, Thangamalai R, Kandasamy K et al (2013). TRPV4 channel activation leads to endothelium‐dependent relaxation mediated by nitric oxide and endothelium‐derived hyperpolarizing factor in rat pulmonary artery. Pharmacol Res 78: 18–27. [DOI] [PubMed] [Google Scholar]
  39. Suresh K, Servinsky L, Reyes J, Baksh S, Undem C, Caterina M et al (2015). Hydrogen peroxide‐induced calcium influx in lung microvascular endothelial cells involves TRPV4. Am J Physiol Lung Cell Mol Physiol 309: L1467–L1477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Suzuki M, Mizuno A, Kodaira K, Imai M (2003). Impaired pressure sensation in mice lacking TRPV4. J Biol Chem 278: 22664–22268. [DOI] [PubMed] [Google Scholar]
  41. Tan GK, Ng JK, Trasti SL, Schul W, Yip G, Alonso S (2010). A non mouse‐adapted dengue virus strain as a new model of severe dengue infection in AG129 mice. PLoS Negl Trop Dis 4: e672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Theiss AL, Vijay‐Kumar M, Obertone TS, Jones DP, Hansen JM, Gewirtz AT et al (2009). Prohibitin is a novel regulator of antioxidant response that attenuates colonic inflammation in mice. Gastroenterology 137: 199–208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Thorneloe KS, Sulpizio AC, Lin Z, Figueroa DJ, Clouse AK, McCafferty GP et al (2008). N‐((1S)‐1‐{[4‐((2S)‐2‐{[(2,4‐dichlorophenyl)sulfonyl]amino}‐3‐hydroxypropanoyl)‐1‐piperazinyl]carbonyl}‐3‐methylbutyl)‐1‐benzothiophene‐2‐carboxamide (GSK1016790A), a novel and potent transient receptor potential vanilloid 4 channel agonist induces urinary bladder contraction and hyperactivity: Part I. J Pharmacol Exp Ther 326: 432–442. [DOI] [PubMed] [Google Scholar]
  44. Tiruppathi C, Minshall RD, Paria BC, Vogel SM, Malik AB (2002). Role of Ca2+ signaling in the regulation of endothelial permeability. Vascul Pharmacol 39: 173–185. [DOI] [PubMed] [Google Scholar]
  45. Tolstanova G, Deng X, French SW, Lungo W, Paunovic B, Khomenko T et al (2012). Early endothelial damage and increased colonic vascular permeability in the development of experimental ulcerative colitis in rats and mice. Lab Invest 92: 9–21. [DOI] [PubMed] [Google Scholar]
  46. Utsumi D, Matsumoto K, Amagase K, Horie S, Kato S (2016). 5‐HT3 receptors promote colonic inflammation via activation of substance P/neurokinin‐1 receptors in dextran sulphate sodium‐induced murine colitis. Br J Pharmacol 173: 1835–1849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Vergnolle N (2014). TRPV4: new therapeutic target for inflammatory bowel diseases. Biochem Pharmacol 89: 157–161. [DOI] [PubMed] [Google Scholar]
  48. Vergnolle N, Cenac N, Altier C, Cellars L, Chapman K, Zamponi GW et al (2010). A role for transient receptor potential vanilloid 4 in tonicity‐induced neurogenic inflammation. Br J Pharmacol 159: 1161–1173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Vriens J, Owsianik G, Fisslthaler B, Suzuki M, Janssens A, Voets T et al (2005). Modulation of the Ca2 permeable cation channel TRPV4 by cytochrome P450 epoxygenases in vascular endothelium. Circ Res 97: 908–915. [DOI] [PubMed] [Google Scholar]
  50. Wandall‐Frostholm C, Dalsgaard T, Bajoriūnas V, Oliván‐Viguera A, Sadda V, Beck L et al (2015). Genetic deficit of KCa 3.1 channels protects against pulmonary circulatory collapse induced by TRPV4 channel activation. Br J Pharmacol 172: 4493–4505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Watanabe H, Vriens J, Prenen J, Droogmans G, Voets T, Nilius B (2003). Anandamide and arachidonic acid use epoxyeicosatrienoic acids to activate TRPV4 channels. Nature 424: 434–438. [DOI] [PubMed] [Google Scholar]
  52. Weidner C, Klede M, Rukwied R, Lischetzki G, Neisius U, Skov PS et al (2000). Acute effects of substance P and calcitonin gene‐related peptide in human skin – a microdialysis study. J Invest Dermatol 115: 1015–1020. [DOI] [PubMed] [Google Scholar]
  53. Willette RN, Bao W, Nerurkar S, Yue TL, Doe CP, Stankus G et al (2008). Systemic activation of the transient receptor potential vanilloid subtype 4 channel causes endothelial failure and circulatory collapse: part 2. J Pharmacol Exp Ther 326: 443–452. [DOI] [PubMed] [Google Scholar]
  54. Wu JC, Yan HC, Chen WT, Chen WH, Wang CJ, Chi YC et al (2008). JNK signaling pathway is required for bFGF‐mediated surface cadherin downregulation on HUVEC. Exp Cell Res 314: 421–429. [DOI] [PubMed] [Google Scholar]
  55. Yamawaki H, Mihara H, Suzuki N, Nishizono H, Uchida K, Watanabe S et al (2014). Role of transient receptor potential vanilloid 4 activation in indomethacin‐induced intestinal damage. Am J Physiol Gastrointest Liver Physiol 307: G33–G40. [DOI] [PubMed] [Google Scholar]
  56. Yasuda M, Kawahara R, Hashimura H, Yamanaka N, Iimori M, Amagase K et al (2011). Dopamine D2–receptor antagonists ameliorate indomethacin‐induced small intestinal ulceration in mice by activating α7 nicotinic acetylcholine receptors. J Pharmacol Sci 116: 274–282. [DOI] [PubMed] [Google Scholar]
  57. Yin J, Michalick L, Tang C, Tabuchi A, Goldenberg N, Dan Q et al (2016). Role of transient receptor potential vanilloid 4 in neutrophil activation and acute lung injury. Am J Respir Cell Mol Biol 54: 370–383. [DOI] [PubMed] [Google Scholar]
  58. Yue Z, Xie J, Yu AS, Stock J, Du J, Yue L (2015). Role of TRP channels in the cardiovascular system. Am J Physiol Heart Circ Physiol 308: H157–H182. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 TRPV4 expression in the colon of bone marrow‐chimeric mice and bone marrow cells. Bone marrow cells harvested from WT or TRPV4 KO mice were transferred to irradiated WT or TRPV4 KO recipient mice, yielding four experimental groups: 1) WT mice receiving WT bone marrow cells (WT → WT), 2) TRPV4 KO mice receiving WT bone marrow cells (WT → KO), 3) WT mice receiving TRPV4 KO bone marrow cells (KO → WT), and 4) TRPV4 KO mice receiving TRPV4 KO bone marrow cells (KO → KO). A) Double‐labelling of TRPV4 (green) and F4/80 (red) in the colon of mice treated with DSS for 7 days. Arrows indicate TRPV4 and F4/80 double‐positive cells. B) Double‐labelling of TRPV4 (green) and F4/80 (red) in bone marrow cells. Scale bars are 50 μm (A) and 10 μm (B).

Click here for additional data file. (9.1MB, tif)

Data S1 Supporting information.

Click here for additional data file. (44KB, doc)

Table S1 Primary antibodies.

Click here for additional data file. (44KB, doc)

Table S2 Secondary antibodies.

Click here for additional data file. (43.5KB, doc)

Articles from British Journal of Pharmacology are provided here courtesy of The British Pharmacological Society

RESOURCES