trpv4 antagonist hc 067047  (Tocris)

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Loss of TRPV4 Cation Channel Inhibition of Macrophage Infiltration and Neovascularization in a Mouse Cornea.

doi: 10.1016/j.labinv.2022.100061

Figure Lengend Snippet: Figure 1. Immunohistochemical localization of TRPV4 in corneal stromal neovascularization induced by central corneal cauterization. Immunohistochemistry detects TRPV4 in a healthy, uninjured corneal epithelium and at day 7 postcauterization at the center of the cornea (A, B). Keratocyte in the stroma was not stained for TRPV4 even at day 7 postinjury (B). (C) Hematoxylin and eosin staining of a day 7 specimen. At day 7 postcauterization, neovascularization (NV) is labeled for cluster of differentiation 31 (CD31) (D) and TRPV4 (E), as observed by using double immunofluorescent staining. (F) A merged picture of panels D and E. TRPV4 is also detected in corneal and conjunctival epithelia, corneal endothelium, conjunctival vessels, scleral fibroblasts, and ciliary body. CD31 is observed in trabecular meshwork cells and conjunctival vessels besides neovascularization. The conjunctival epithelium is rolled up. (G) A similar area of the mouse limbal region in a day 7 specimen. Arrows indicate conjunctival vessels, and arrowheads indicate scleral fibroblasts. Bar ¼ 100 mm. CD31, cluster of differentiation 31; NV, neovascularization; TM, trabecular meshwork; TRPV4, transient receptor potential vanilloid 4.

Article Snippet: We, therefore, performed the 12-hour culture experiments in a medium supplemented with sulforaphane (15 mM, for positive control25), TRPV4 antagonist HC-067047 (0.1 mM,1 mM, or 10 mM; TOCRIS), or control medium.

Techniques: Immunohistochemical staining, Immunohistochemistry, Staining, Labeling

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Loss of TRPV4 Cation Channel Inhibition of Macrophage Infiltration and Neovascularization in a Mouse Cornea.

doi: 10.1016/j.labinv.2022.100061

Figure Lengend Snippet: Figure 2. Neovascularization in the peripheral cornea from the limbus in response to cauterization at the cornea center. The uninjured cornea of either WT (A) or a TRPV4-null (knockout) (C) mouse lacks neovascularization in the peripheral area of the cornea as observed by suing CD31 immunodetection in a flat-mounted specimen. At day 7 postcauterization, inwardly elongating new vessels from the limbus were detected in both genotypes of mice. The observation suggested that the loss of TRPV4 (D) might suppress their elongation as compared to a WT mouse (B). Bar ¼ 1000 mm. CD31, cluster of differentiation 31; KO, knockout; TRPV4, transient receptor potential vanilloid 4; WT, wild-type.

Article Snippet: We, therefore, performed the 12-hour culture experiments in a medium supplemented with sulforaphane (15 mM, for positive control25), TRPV4 antagonist HC-067047 (0.1 mM,1 mM, or 10 mM; TOCRIS), or control medium.

Techniques: Knock-Out, Immunodetection

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Loss of TRPV4 Cation Channel Inhibition of Macrophage Infiltration and Neovascularization in a Mouse Cornea.

doi: 10.1016/j.labinv.2022.100061

Figure Lengend Snippet: Figure 3. Loss of TRPV4 attenuates corneal neovascularization in response to cauterization at the cornea center. (A) Histologic examination indicated that the distance between the tip (white arrowheads) of CD31-immunostained neovascularization in corneal stroma and the limbus (black arrowheads) seems less in TRPV4-null (KO) mice than in the WT mice at days 3, 7, and 14 7. (B) The distance between the neovascularization tip and the limbus was significantly less in KO mice (DeF) than in WT mice (AeC) on days 3, 7, and 14. Bar ¼ 100 mm. **P < .01 and *P < .05. CD31, cluster of differentiation 31; KO, knockout; TRPV4, transient receptor potential vanilloid 4; WT, wild-type.

Article Snippet: We, therefore, performed the 12-hour culture experiments in a medium supplemented with sulforaphane (15 mM, for positive control25), TRPV4 antagonist HC-067047 (0.1 mM,1 mM, or 10 mM; TOCRIS), or control medium.

Techniques: Knock-Out

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Loss of TRPV4 Cation Channel Inhibition of Macrophage Infiltration and Neovascularization in a Mouse Cornea.

doi: 10.1016/j.labinv.2022.100061

Figure Lengend Snippet: Figure 4. Loss of TRPV4 attenuates the infiltration of macrophages in the cauterized cornea but does not affect myofibroblast transformation. (A) Increases in F4/80 mRNA expression level as determined by real-time reverse-transcription PCR were indicative of macrophage population in the cornea in a healthy, uninjured, WT stroma, which is more marked as compared with a TRPV4-null tissue. (A) Macrophage infiltration in the centrally cauterized cornea is reduced by the loss of the TRPV4 gene at day 3. In immunohistochemical histology, F4/80-labeled macrophages were detected in the stroma at the site of cauterization in a WT cornea on day 7. The number of cells seemed to decrease on day 14. (B) On the other hand, F4/80-positive cells were not seen under immunohistochemistry level at days 7 and 14. At day 3 postcauterization, the total expression level of aSMA, a myofibroblast marker, was not significantly altered by the loss of TRPV4 (C). *P <.05. aSMA, a-smooth muscle actin; mRNA, messenger RNA; n.s., not significant; TRPV4, transient receptor potential vanilloid 4; WT, wild-type.

Article Snippet: We, therefore, performed the 12-hour culture experiments in a medium supplemented with sulforaphane (15 mM, for positive control25), TRPV4 antagonist HC-067047 (0.1 mM,1 mM, or 10 mM; TOCRIS), or control medium.

Techniques: Transformation Assay, Expressing, Reverse Transcription, Immunohistochemical staining, Labeling, Immunohistochemistry, Marker

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Loss of TRPV4 Cation Channel Inhibition of Macrophage Infiltration and Neovascularization in a Mouse Cornea.

doi: 10.1016/j.labinv.2022.100061

Figure Lengend Snippet: Figure 5. Loss of TRPV4 suppresses the tissue expression of VEGF-A. The mRNA expression level of VEGF-A was suppressed by the loss of TRPV4 (A) both in an uninjured cornea and on day 3 postcauterization. Expression of IL-6, TGFb1, NGF, MCP-1, and TNFa was not statistically significantly affected by the loss of TRPV4 in mouse corneas (B-F). **P < .01, *P < .05. IL-6, interleukin 6; MCP-1, macrophage chemoattractant protein 1; mRNA, messenger RNA; NGF, nerve growth factor; n.s., not significant; TGFb1, transforming growth factor-b1; TNFa, tumor necrosis factor a; TRPV4, transient receptor potential vanilloid 4; VEGF-A, vascular endothelial growth factor A.

Article Snippet: We, therefore, performed the 12-hour culture experiments in a medium supplemented with sulforaphane (15 mM, for positive control25), TRPV4 antagonist HC-067047 (0.1 mM,1 mM, or 10 mM; TOCRIS), or control medium.

Techniques: Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Loss of TRPV4 Cation Channel Inhibition of Macrophage Infiltration and Neovascularization in a Mouse Cornea.

doi: 10.1016/j.labinv.2022.100061

Figure Lengend Snippet: Figure 6. Supplementation of a TRPV4 antagonist inhibits VEGF-Aeinduced cultured HUVEC tube formation. (A) Fluorescein-labeled images of tube-like tissue formation by HUVECs in vitro. Supplementation of sulforaphane (an autophagy inhibitor, a positive [inhibitory] control) effectively suppressed tube-like structure formation. HC-067047, a TRPV4 antagonist, administration suppressed tube-like tissue formation. (B) Quantified data of the length of tube-like structure formation. Bar ¼ 500 mm. **P < .01. HUVEC, human umbilical vein endothelial cell; TRPV4, transient receptor potential vanilloid 4; VEGF-A, vascular endothelial growth factor A.

Article Snippet: We, therefore, performed the 12-hour culture experiments in a medium supplemented with sulforaphane (15 mM, for positive control25), TRPV4 antagonist HC-067047 (0.1 mM,1 mM, or 10 mM; TOCRIS), or control medium.

Techniques: Cell Culture, Labeling, In Vitro, Control

Journal: eLife

Article Title: TRPV4 activation by TGFβ2 enhances cellular contractility and drives ocular hypertension

doi: 10.7554/eLife.104894

Figure Lengend Snippet: ( A, B ) Five-day TGFβ2 treatment (1 ng/ml) significantly altered expression of TGFβ pathway effectors, cytoskeletal machinery, and canonical fibrotic markers. ( C ) TGFβ2 treatment significantly increased TRPV4 and PIEZO1 expression, but not TREK1 and TRPC1 expression. Mean ± SEM shown. N = 4–8 experiments, each gene tested in 3–7 different pTM strains (see ). Two-tailed one-sample t -test of TGFβ2-induced gene expression levels as a percent of control samples. ( D ) Isolation of membrane proteins from two separate pooled pTM samples suggests TGFβ2 treatment drives increased TRPV4 membrane insertion. N = 2 independent pooled samples, 3 pTM strains were pooled per sample. *p < 0.05, **p < 0.01. Figure 1—source data 1. Uncropped images of the membrane and HRP-signal for the western blots shown in (labelled). Figure 1—source data 2. Uncropped images of the membrane and HRP-signal for the western blots shown in .

Article Snippet: The TRPV4 antagonist HC-067047 (HC-06) was purchased from Millipore-Sigma (Burlington, MA) or Cayman Biotech (Ann Arbor, MI) and dissolved in DMSO at 20 mM.

Techniques: Expressing, Two Tailed Test, Gene Expression, Control, Isolation, Membrane, Western Blot

Journal: eLife

Article Title: TRPV4 activation by TGFβ2 enhances cellular contractility and drives ocular hypertension

doi: 10.7554/eLife.104894

Figure Lengend Snippet: ( A ) Five-day TGFβ2 treatment (1 ng/ml) increased TRPV4 agonist-induced (GSK101, 10 nM) Ca 2+ influx in primary trabecular meshwork (pTM) cells compared to serum-free media alone treated cells tested on the same day ( N = 5 pTM strains, n = 3–5 slides/condition/day, individual data points over mean ± SEM). Two-tailed one-sample t -test of TGFβ2-treated cell average GSK101 response as a percent of control samples from the same pTM strain on the same day. ( B ) Violin plots showing the distribution of GSK101-induced Ca 2+ responses for each pTM strain tested in A. Thick dashed line indicates mean, while light dashed line indicates quartiles. ( C ) Representative traces showing TRPV4 agonist-induced Ca 2+ influx (seen as an increase in F 340 / F 380 ) in pTM (mean ± SEM of 4 representative cells/group), alongside example Fura-2-loaded pTM cells before ( i ), during ( ii ), and after ( iii ) GSK101 application. Scale bar = 50 µm. **p < 0.01.

Article Snippet: The TRPV4 antagonist HC-067047 (HC-06) was purchased from Millipore-Sigma (Burlington, MA) or Cayman Biotech (Ann Arbor, MI) and dissolved in DMSO at 20 mM.

Techniques: Two Tailed Test, Control

Journal: eLife

Article Title: TRPV4 activation by TGFβ2 enhances cellular contractility and drives ocular hypertension

doi: 10.7554/eLife.104894

Figure Lengend Snippet: ( A ) TGFβ2 treatments for 24 hr at 1 ng/ml ( N = 6 pTM strains, n = 3–5 slides/condition/day) or 5 ng/ml ( N = 5 pTM strains, n = 3–5 slides/condition/day) did not show potentiation of GSK101-evoked TRPV4 Ca 2+ influx and were significantly lower than cells treated with TGFβ2 for 5 days at 1 ng/ml (5 days TGFβ2 results from ). Individual data points over mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons test, statistics for individual 1 day treatment groups compared to control groups shown in . ( B ) Representative traces for GSK101 response following 24 hr TGFβ2 treatment, traces show mean ± SEM of 3–4 cells. ( C ) Average current density in response to GSK101 (24 hr control: n = 11 cells, 24 hr TGFβ2: n = 10 cells) shows generally increased current in TGFβ2-treated cells. Data show mean ± SEM ( D, E ). Violin plots of individual cell strains shown in A . Thick dashed line indicates mean, while light dashed line indicates quartiles. ** p < 0.01, *** p < 0.001.

Article Snippet: The TRPV4 antagonist HC-067047 (HC-06) was purchased from Millipore-Sigma (Burlington, MA) or Cayman Biotech (Ann Arbor, MI) and dissolved in DMSO at 20 mM.

Techniques: Control

Journal: eLife

Article Title: TRPV4 activation by TGFβ2 enhances cellular contractility and drives ocular hypertension

doi: 10.7554/eLife.104894

Figure Lengend Snippet: ( A ) Intravitreal injection of LV-TGFβ2 (Week 1), but not LV-Control, elevates intraocular pressure (IOP) in WT mice ( N = 5 eyes/group) as early as 1-week post-injection. Injection of TRPV4 antagonist HC-06, but not PBS, produced multiday IOP reduction in LV-TGFβ2-treated eyes. HC-06 and PBS injections did not affect IOP in LV-Control injected eyes. Two-way ANOVA with Bonferroni post hoc analysis ( B ) Direct comparison of the results of PBS and HC-06 injections in the eyes shown in A . Two-way ANOVA with Bonferroni post hoc analysis. ( C ) Intravitreal injection of LV-TGFβ2 in Trpv4 −/− mice ( N = 6 eyes/group) resulted in only mild OHT; plotted against WT eyes at matching timepoints (3 WT cohorts including the 5 WT eyes shown in A, B, N = 8–15 eyes/group). ( D ) Statistical comparison of the IOP values shown in C . The IOP in LV-TGFβ2 WT eyes was significantly elevated compared to the LV-TGFβ2 Trpv4 −/− eyes from 2 weeks post-injection. LV-Control injected eyes in WT or Trpv4 −/− eyes remain close to the baseline value and are not significantly different. Two-way ANOVA with Bonferroni post hoc analysis. ( A, C ) shows mean ± SEM. Data in ( B, D ) show individual data points over mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Figure 5—source data 1. Source data for Lv-Control IOP and Lv-TFFb2 cohorts treated with HC-06. Figure 5—source data 2. Source data for IOP data from WT and Trpv4 KO eyes treated with TGFβ2.

Article Snippet: The TRPV4 antagonist HC-067047 (HC-06) was purchased from Millipore-Sigma (Burlington, MA) or Cayman Biotech (Ann Arbor, MI) and dissolved in DMSO at 20 mM.

Techniques: Injection, Control, Produced, Comparison

Journal: eLife

Article Title: TRPV4 activation by TGFβ2 enhances cellular contractility and drives ocular hypertension

doi: 10.7554/eLife.104894

Figure Lengend Snippet: Intraocular pressure (IOP) in LV-TGFβ2-injected eyes was significantly elevated compared to both LV-Ctrl injected WT and Trpv4 −/− eyes, as well as LV-TGFβ2-injected Trpv4 −/− eyes ( N = 6 eyes/condition).

Article Snippet: The TRPV4 antagonist HC-067047 (HC-06) was purchased from Millipore-Sigma (Burlington, MA) or Cayman Biotech (Ann Arbor, MI) and dissolved in DMSO at 20 mM.

Techniques: Injection

Journal: eLife

Article Title: TRPV4 activation by TGFβ2 enhances cellular contractility and drives ocular hypertension

doi: 10.7554/eLife.104894

Figure Lengend Snippet: Chronic exposure to TGFβ2 induces upregulation of functional TRPV4 channels alongside the autoinhibitory canonical modulator SMAD7. TRPV4-mediated Ca 2+ influx, canonical, and non-canonical TGFβ2 signaling stimulate the Rho/ROCK pathway to augment cytoskeletal contractility and stimulate extracellular matrix (ECM) release. Actomyosin contractility promotes outflow resistance and drives OHT and underpins a vicious feedforward TRPV4-dependent loop that maintains OHT. This figure was created using BioRender.com .

Article Snippet: The TRPV4 antagonist HC-067047 (HC-06) was purchased from Millipore-Sigma (Burlington, MA) or Cayman Biotech (Ann Arbor, MI) and dissolved in DMSO at 20 mM.

Techniques: Functional Assay

Journal: Molecules

Article Title: Establishing an ANO1-Based Cell Model for High-Throughput Screening Targeting TRPV4 Regulators

doi: 10.3390/molecules29051036

Figure Lengend Snippet: Schematic diagram of the cell model under the action of TRPV4 channel modulators. ( A ) YFP stands for Yellow Fluorescent Protein. When the TRPV4 agonist is present outside the cell, it stimulates the opening of the TRPV4 channel, which causes a large inward flow of calcium ions from outside the cell. The increased intracellular calcium ion concentration activates the opening of ANO1 channels, which translocate large amounts of halide ions into the cell, thereby quenching the fluorescent signal of the YFP- H148Q/I152L fluorescent protein in the cytoplasm of the cell. ( B ) Inhibition of channel opening in the presence of a TRPV4 inhibitor. Even in the presence of an agonist in the external fluid, no opening of the TRPV4 was induced and the fluorescence intensity in the cytoplasm of the cell was unchanged.

Article Snippet: F-12 nutrient media, enhanced chemiluminescent (ECL), Eact (ANO1 activator), Niflumic acid (NFA, a Cl − channel blocker), 4α-phorbol 12,13-didecanoate (4αPDD, a TRPV4 agonist), GSK1016790A (TRPV4 agonist), RN-1747 (TRPV4 agonist), and HC 067047 (TRPV4 antagonist) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Concentration Assay, Inhibition, Fluorescence

Journal: Molecules

Article Title: Establishing an ANO1-Based Cell Model for High-Throughput Screening Targeting TRPV4 Regulators

doi: 10.3390/molecules29051036

Figure Lengend Snippet: Results of endogenous expression of the TRPV4 protein in FRT cells. ( A1 ) Electrophoresis results. qRT-PCR results showing specific bands at 455 bp, 395 bp, and 260 bp for Trpv41, Trpv42, and Actb (β-actin), respectively. ( A2 ) Sequencing results of the Trpv4 gene from FRT cells, where the vertical line shows the intron. ( A3 ) Results of searching with the sequence from A2 in the GenBank database, the similarity to the sequenced Trpv4 is 100%. ( B ) Results of siRNA knockdown of TRPV4, ** p < 0.01. ( C1 ) Western blotting analysis results. The β-actin relative band is at 42 kDa and the TRPV4 protein band is at 98 kDa. ( C2 ) The intensity ratio of the bands in ( C1 ).

Article Snippet: F-12 nutrient media, enhanced chemiluminescent (ECL), Eact (ANO1 activator), Niflumic acid (NFA, a Cl − channel blocker), 4α-phorbol 12,13-didecanoate (4αPDD, a TRPV4 agonist), GSK1016790A (TRPV4 agonist), RN-1747 (TRPV4 agonist), and HC 067047 (TRPV4 antagonist) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Expressing, Electrophoresis, Quantitative RT-PCR, Sequencing, Western Blot

Journal: Molecules

Article Title: Establishing an ANO1-Based Cell Model for High-Throughput Screening Targeting TRPV4 Regulators

doi: 10.3390/molecules29051036

Figure Lengend Snippet: Electrophysiological properties of TRPV4. ( A ) Recording of the −100 mV to 100 mV cell membrane current after stable clamping of FRT cells. ( B ) Recording of the −100 mV to 100 mV cell membrane current after the addition of 100 nM GSK1016790A. ( C ) Recording of the −100 mV to 100 mV cell membrane current after the addition of 10 μM HC-067047, followed by the addition of 100 nM GSK1016790A. ( D ) Elution of the inhibitor followed by the addition of 100 nM GSK1016790A to record the −100 mV to 100 mV cell membrane currents. ( E ) Analysis of mean cell currents for each group using GraphPad 9.3.0 software (Mean ± SD, n = 3), ** p < 0.01. ( F ) Current–voltage relationship curves.

Article Snippet: F-12 nutrient media, enhanced chemiluminescent (ECL), Eact (ANO1 activator), Niflumic acid (NFA, a Cl − channel blocker), 4α-phorbol 12,13-didecanoate (4αPDD, a TRPV4 agonist), GSK1016790A (TRPV4 agonist), RN-1747 (TRPV4 agonist), and HC 067047 (TRPV4 antagonist) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Membrane, Software

Journal: Molecules

Article Title: Establishing an ANO1-Based Cell Model for High-Throughput Screening Targeting TRPV4 Regulators

doi: 10.3390/molecules29051036

Figure Lengend Snippet: Validation of the TRPV4 cell model. ( A ) Validation results of the TRPV4 cell model, ANO1 channel performance. ( B ) Change in the relative fluorescence intensity of cells caused by the TRPV4 cell model, ANO1 channel (Mean ± SD, n = 3), * p < 0.001, ** p < 0.001, *** p < 0.001. ( C ) TRPV4 cell model, TRPV4 channel performance validation. ( D ) TRPV4 cell model, TRPV4 channel-induced changes in relative cellular fluorescence intensity (Mean ± SD, n = 3), ** p < 0.01.

Article Snippet: F-12 nutrient media, enhanced chemiluminescent (ECL), Eact (ANO1 activator), Niflumic acid (NFA, a Cl − channel blocker), 4α-phorbol 12,13-didecanoate (4αPDD, a TRPV4 agonist), GSK1016790A (TRPV4 agonist), RN-1747 (TRPV4 agonist), and HC 067047 (TRPV4 antagonist) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Fluorescence

Journal: Molecules

Article Title: Establishing an ANO1-Based Cell Model for High-Throughput Screening Targeting TRPV4 Regulators

doi: 10.3390/molecules29051036

Figure Lengend Snippet: Functional validation results of TRPV4 cell model. ( A ) Effect of temperature on the TRPV4 channels assessed using flow cytometry. ( B ) Analysis of the mean fluorescence intensity per group in group A (Mean ± SD, n = 3), ** p < 0.001, *** p < 0.001. ( C ) Dose-dependent relationship of TRPV4 to the agonists. ( D ) Dose-dependent relationship of TRPV4 to the inhibitors.

Article Snippet: F-12 nutrient media, enhanced chemiluminescent (ECL), Eact (ANO1 activator), Niflumic acid (NFA, a Cl − channel blocker), 4α-phorbol 12,13-didecanoate (4αPDD, a TRPV4 agonist), GSK1016790A (TRPV4 agonist), RN-1747 (TRPV4 agonist), and HC 067047 (TRPV4 antagonist) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Functional Assay, Flow Cytometry, Fluorescence

Journal: Molecules

Article Title: Establishing an ANO1-Based Cell Model for High-Throughput Screening Targeting TRPV4 Regulators

doi: 10.3390/molecules29051036

Figure Lengend Snippet: Results of the intracellular calcium ion concentration in the TRPV4 cell model. ( A ) The intracellular calcium ion concentration and GSK1016790A were in a dose-dependent relationship. ( B ) Relative change in the fluorescence values for FRT cells shows a dose-dependent relationship with GSK1016790A. ( C ) The intracellular calcium ion concentration and relative change in the fluorescence values in FRT cells.

Article Snippet: F-12 nutrient media, enhanced chemiluminescent (ECL), Eact (ANO1 activator), Niflumic acid (NFA, a Cl − channel blocker), 4α-phorbol 12,13-didecanoate (4αPDD, a TRPV4 agonist), GSK1016790A (TRPV4 agonist), RN-1747 (TRPV4 agonist), and HC 067047 (TRPV4 antagonist) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Concentration Assay, Fluorescence

Journal: Molecules

Article Title: Establishing an ANO1-Based Cell Model for High-Throughput Screening Targeting TRPV4 Regulators

doi: 10.3390/molecules29051036

Figure Lengend Snippet: Primer sequence.

Article Snippet: F-12 nutrient media, enhanced chemiluminescent (ECL), Eact (ANO1 activator), Niflumic acid (NFA, a Cl − channel blocker), 4α-phorbol 12,13-didecanoate (4αPDD, a TRPV4 agonist), GSK1016790A (TRPV4 agonist), RN-1747 (TRPV4 agonist), and HC 067047 (TRPV4 antagonist) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Sequencing

Journal: Stroke and Vascular Neurology

Article Title: Delta opioid peptide [D-ala2, D-leu5]-Enkephalin’s ability to enhance mitophagy via TRPV4 to relieve ischemia/reperfusion injury in brain microvascular endothelial cells

doi: 10.1136/svn-2023-003080

Figure Lengend Snippet: The sequences of siRNA targeting TRPV4 and scrambled siRNA

Article Snippet: The δOR agonist, DADLE (E7131, Sigma-Aldrich), the TRPV4 agonist, GSK1016790A (HY-19608, MCE) and the TRPV4 antagonist, HC-067047 (HY-100208, MCE) were dissolved in DMSO.

Techniques:

Journal: Stroke and Vascular Neurology

Article Title: Delta opioid peptide [D-ala2, D-leu5]-Enkephalin’s ability to enhance mitophagy via TRPV4 to relieve ischemia/reperfusion injury in brain microvascular endothelial cells

doi: 10.1136/svn-2023-003080

Figure Lengend Snippet: DADLE significantly ameliorates the reduction in TRPV4 expression in primary cultured BMECs caused by OGD/R. ( A ) Expressive detection of δORs in BMECs. Inverted microscope magnification: 100×, scale bar=100 µm; 200×, scale bar=50 µm. Statistical bar shows the ratio of the area of positive dual-staining signal to the area of cells (n=4). ( B ) Localisation of δORs. Laser confocal oil microscope magnification: 600×, scale bar=20 µm (n=3). ( C ) Volcano plot of next-generation high-throughput sequencing (RNAseq): red points represent upregulated genes, and green points represent downregulated genes. ( D ) The Venn diagram shows 43 intersecting genes of the two volcano plots in panel C, including TRPV4. ( E ) Western blotting validated the TRPV4 expressive change (n=4). ( F ) Western blotting examined the effect of different treatments on the expression level of δOR (n=5). Data are presented as mean±SEM and ns indicates no statistical significance, *p<0.05. δOR, δ opioid receptor; BMECs, brain microvascular endothelial cells; DADLE, [D-ala2, D-leu5]-Enkephalin; OGD/R, oxygen-glucose deprivation/reoxygenation; PBS, phosphate-buffered saline; TRPV4, transient receptor potential subfamily V member 4; vWF, von willebrand factor; DAPI, 4',6-diamidino-2-phenylindole.

Article Snippet: The δOR agonist, DADLE (E7131, Sigma-Aldrich), the TRPV4 agonist, GSK1016790A (HY-19608, MCE) and the TRPV4 antagonist, HC-067047 (HY-100208, MCE) were dissolved in DMSO.

Techniques: Expressing, Cell Culture, Inverted Microscopy, Staining, Microscopy, Next-Generation Sequencing, Western Blot, Saline

Journal: Stroke and Vascular Neurology

Article Title: Delta opioid peptide [D-ala2, D-leu5]-Enkephalin’s ability to enhance mitophagy via TRPV4 to relieve ischemia/reperfusion injury in brain microvascular endothelial cells

doi: 10.1136/svn-2023-003080

Figure Lengend Snippet: DADLE (5 nM) increases TRPV4-mediated calcium influx in BMECs affected by OGD/R injury. ( A ) Calcium imaging assay was used to detect the signal of calcium ion influx mediated by TRPV4 activated with 50 nM GSK1016790A (n=5). ( B ) In BMECs, after depletion of calcium stores with 2 µM TG, extracellular calcium at 2 mM was provided to detect the signal of SOCE. Concurrent addition of 5 µM HC067047 acted as a TRPV4 inhibitor (n=6). Data were presented as mean±SEM. **p<0.01, ***p<0.001. BMECs, brain microvascular endothelial cells; DADLE, [D-ala2, D-leu5]-Enkephalin; OGD/R, oxygen-glucose deprivation/reoxygenation; SOCE, store-operated Ca 2+ entry; TRPV4, transient receptor potential subfamily V member 4.

Article Snippet: The δOR agonist, DADLE (E7131, Sigma-Aldrich), the TRPV4 agonist, GSK1016790A (HY-19608, MCE) and the TRPV4 antagonist, HC-067047 (HY-100208, MCE) were dissolved in DMSO.

Techniques: Imaging

Journal: Stroke and Vascular Neurology

Article Title: Delta opioid peptide [D-ala2, D-leu5]-Enkephalin’s ability to enhance mitophagy via TRPV4 to relieve ischemia/reperfusion injury in brain microvascular endothelial cells

doi: 10.1136/svn-2023-003080

Figure Lengend Snippet: DADLE (5 nM) elevates mitophagy in BMECs under OGD/R via TRPV4. ( A ) Expression levels of autophagy-related proteins Beclin-1 (n=3), p62 (n=8), LC3-II/I (n=3), PINK1 (n=4) and Parkin (n=8) were examined using western blot; 5 µM HC067047 were used to inhibit TRPV4. ( B ) Autophagosome conformation within BMECs was visualised using TEM (n=3). Images taken at increasing magnifications are arranged from top to bottom, with magnifications of 2600×, scale bar=5 µm; 13500×, scale bar=500 nm; 36000×, scale bar=200 nm. Data are presented as mean±SEM. *p<0.05, **p<0.01, ***p<0.001. BMECs, brain microvascular endothelial cells; DADLE, [D-ala2, D-leu5]-Enkephalin; OGD/R, oxygen-glucose deprivation/reoxygenation; TEM, transmission electron microscopy; TRPV4, transient receptor potential subfamily V member 4.

Article Snippet: The δOR agonist, DADLE (E7131, Sigma-Aldrich), the TRPV4 agonist, GSK1016790A (HY-19608, MCE) and the TRPV4 antagonist, HC-067047 (HY-100208, MCE) were dissolved in DMSO.

Techniques: Expressing, Western Blot, Transmission Assay, Electron Microscopy

Journal: Stroke and Vascular Neurology

Article Title: Delta opioid peptide [D-ala2, D-leu5]-Enkephalin’s ability to enhance mitophagy via TRPV4 to relieve ischemia/reperfusion injury in brain microvascular endothelial cells

doi: 10.1136/svn-2023-003080

Figure Lengend Snippet: The use of 5 µM HC067047 inhibits the effects of 5 nM DADLE on the mitochondria of BMECs under OGD/R conditions. ( A ) Changes in mitochondrial membrane potential levels following TRPV4 inhibition were evaluated using JC-1 staining under confocal microscopy (n=5). ( B ) ATP synthesis of BMECs after TRPV4 inhibition was examined (n=6). ( C ) Relative cell viability after TRPV4 inhibition was assessed via CCK8 assay (n=6). Data are expressed as mean±SEM. *p<0.05, **p<0.01, ***p<0.001. BMECs, brain microvascular endothelial cells; DADLE, [D-ala2, D-leu5]-Enkephalin; OGD/R, oxygen-glucose deprivation/reoxygenation; TRPV4, transient receptor potential subfamily V member 4.

Article Snippet: The δOR agonist, DADLE (E7131, Sigma-Aldrich), the TRPV4 agonist, GSK1016790A (HY-19608, MCE) and the TRPV4 antagonist, HC-067047 (HY-100208, MCE) were dissolved in DMSO.

Techniques: Membrane, Inhibition, Staining, Confocal Microscopy, CCK-8 Assay

Journal: Stroke and Vascular Neurology

Article Title: Delta opioid peptide [D-ala2, D-leu5]-Enkephalin’s ability to enhance mitophagy via TRPV4 to relieve ischemia/reperfusion injury in brain microvascular endothelial cells

doi: 10.1136/svn-2023-003080

Figure Lengend Snippet: Interference with TRPV4 mRNA synthesis using siRNA. ( A ) The interference effect of siRNA on TRPV4 was measured by western blotting (n=4). ( B ) Determination of the expression of the autophagyrelated proteins Beclin-1 (n=4), p62 (n=7), LC3-II/I (n=6), PINK1 (n=3) and Parkin (n=3) after TRPV4 interference. ( C ) Changes in the level of mitochondrial membrane potential in BMECs after TRPV4 interference (n=9). ( D ) The synthesis of ATP in BMEC cells was examined after TRPV4 interference (n=6). ( E ) Relative cell viability after TRPV4 interference (n=4). Data are expressed as mean±SEM. *p<0.05, **p<0.01, ***p<0.001. BMECs, brain microvascular endothelial cells; DADLE, [D-ala2, D-leu5]-Enkephalin; mRNA, messenger RNA; OGD/R, oxygen-glucose deprivation/reoxygenation; TRPV4, transient receptor potential subfamily V member 4.

Article Snippet: The δOR agonist, DADLE (E7131, Sigma-Aldrich), the TRPV4 agonist, GSK1016790A (HY-19608, MCE) and the TRPV4 antagonist, HC-067047 (HY-100208, MCE) were dissolved in DMSO.

Techniques: Western Blot, Expressing, Membrane

Journal: Stroke and Vascular Neurology

Article Title: Delta opioid peptide [D-ala2, D-leu5]-Enkephalin’s ability to enhance mitophagy via TRPV4 to relieve ischemia/reperfusion injury in brain microvascular endothelial cells

doi: 10.1136/svn-2023-003080

Figure Lengend Snippet: Schematic draw (Created in BioRender: Scientific Image and Illustration Software | BioRender). BMECs were extracted from Sprague-Dawley rats and subjected to the OGD/R model to simulate the I/R process. When the cells were treated with 5 nM DADLE, the expression of TRPV4 protein increased, and TRPV4-mediated calcium influx was enhanced, thereby increasing the level of mitophagy, the mitochondrial membrane potential and ATP synthesis, all while reducing I/R injury and promoting cell survival. The above protective effects of DADLE can be blocked through the addition of a TRPV4 inhibitor (HC067047) or RNAi TRPV4. BMECs, brain microvascular endothelial cells; DADLE, [D-ala2, D-leu5]-Enkephalin; I/R, ischaemia/reperfusion; OGD/R, oxygen-glucose deprivation/reoxygenation; TRPV4, transient receptor potential subfamily V member 4.

Article Snippet: The δOR agonist, DADLE (E7131, Sigma-Aldrich), the TRPV4 agonist, GSK1016790A (HY-19608, MCE) and the TRPV4 antagonist, HC-067047 (HY-100208, MCE) were dissolved in DMSO.

Techniques: Software, Expressing, Membrane