Small molecule compound M12 reduces vascular permeability in obese mice via blocking endothelial TRPV4-Nox2 interaction

Abstract

Transient receptor potential channel TRPV4 and nicotinamide adenine dinucleotide phosphate oxidase (Nox2) are involved in oxidative stress that increases endothelial permeability. It has been shown that obesity enhances the physical association of TRPV4 and Nox2, but the role of TRPV4-Nox2 association in obesity has not been clarified. In this study we investigated the function of TRPV4-Nox2 complex in reducing oxidative stress and regulating abnormal vascular permeability in obesity. Obesity was induced in mice by feeding a high-fat diet (HFD) for 14 weeks. The physical interaction between TRPV4 and Nox2 was measured using FRET, co-immunoprecipitation and GST pull-down assays. The functional interaction was measured by rhodamine phalloidin, CM-H₂DCFDA in vitro, the fluorescent dye dihydroethidium (DHE) staining assay, and the Evans blue permeability assay in vivo. We demonstrated that TRPV4 physically and functionally associated with Nox2, and this physical association was enhanced in aorta of obese mice. Furthermore, we showed that interrupting TRPV4-Nox2 coupling by TRPV4 knockout, or by treatment with a specific Nox2 inhibitor Nox2 dstat or a specific TRPV4 inhibitor HC067046 significantly attenuated obesity-induced ROS overproduction in aortic endothelial cells, and reversed the abnormal endothelial cytoskeletal structure. In order to discover small molecules disrupting the over-coupling of TPRV4 and Nox2 in obesity, we performed molecular docking analysis and found that compound M12 modulated TRPV4-Nox2 association, reduced ROS production, and finally reversed disruption of the vascular barrier in obesity. Together, this study, for the first time, provides evidence for the TRPV4 physically interacting with Nox2. TRPV4-Nox2 complex is a potential drug target in improving oxidative stress and disruption of the vascular barrier in obesity. Compound M12 targeting TRPV4-Nox2 complex can improve vascular barrier function in obesity.

Keywords: Compound M12; Nox2; TPRV4; obesity; oxidant stress; vascular permeability.

Fig. 1. The physical association between TRPV4 and Nox2 is enhanced in obesity.

a Left, co-Immunoprecipitation of TRPV4 and Nox2 in primary aorta endothelial cells; right, band intensities normalized to TRPV4 (n = 7 mice; ^*P = 0.0070 vs. ND; Student’s t test and Mann–Whitney test; ND, Wild-type C57BL/6J mice fed with normal diet; HFD, Wild-type C57BL/6J mice fed with high-fat diet). b Representative fluorescence resonance energy transfer (FRET) images of aorta sections from ND and HFD mice (dashed white lines, autofluorescence of elastin; scale bar, 100 μm); (c) Quantification of FRET efficiency of aorta sections from ND (n = 9) and HFD (n = 9) mice (^*P = 0.0025 vs. ND, Student’s t test, unpaired two-tailed t test). d Direct interaction between TRPV4 and Nox2 was demonstrated by in vitro GST pull-down assay. All data are shown as the mean ± SEM.

Fig. 2. Functional role of TRPV4 and Nox2 in ECs in obesity.

a 5,6-Chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA) staining results showing the GSK (100 nM)-induced reactive oxygen species (ROS) in ND aortic ECs and TRPV4-KO aortic ECs (n = 8, ^*P < 0.05 vs. Ctrl, ^#P < 0.05 vs. GSK, one-way ANOVA, Dunnett’s multiple comparisons test). b Statistics of CM-H₂DCFDA fluorescence intensity in mouse aortic ECs isolated from ND mice, HFD mice with or without Nox2 dstat and TRPV4-KO mice (n = 12, ^*P < 0.05 vs. ND, ^#P < 0.05 vs. HFD, one-way ANOVA, Tukey’s multiple comparisons test). c, d Relative mRNA level of VE-cadherin and ICAM-1 in ND, HFD, TRPV4-KO aortic ECs and HFD aortic ECs pretreated with Nox2 dstat (n = 6; ^*P < 0.05 vs. ND, ^#P < 0.05 vs. HFD, Student’s t test, Paired t test). e Representative photomicrographs of rhodamine phalloidin staining and statistical results of F-actin length (scale bars, 100 μm), normalized to ND, (n = 10 mice; ^#P < 0.05 vs. HFD, ^*P < 0.05 vs. ND; one-way ANOVA, Tukey’s multiple comparisons test). f Results of FITC-dextran staining assays showing permeability of ND, HFD, TRPV4-KO aortic ECs and HFD aortic ECs pretreated with Nox2 dstat (n = 8 mice; ^*P < 0.05 vs. ND, ^#P < 0.05 vs. HFD, one-way ANOVA, Tukey’s multiple comparisons test). All data are shown as the mean ± SEM.

Fig. 3. TRPV4 is involved in the regulation of ROS production and vascular permeability in diet-induced obesity mouse model.

a Representative photomicrographs of rhodamine phalloidin staining in arterial sections from ND, HFD, TRPV4 KO-ND, TRPV4 KO-HFD mice (scale bar, 50 μm). b Right, representative photomicrographs of dihydroethidium (DHE) staining in arterial sections from ND, HFD, TRPV4 KO-ND, TRPV4 KO-HFD mice; left, analysis of DHE fluorescence intensity normalized to the value in control aorta (n = 6–7 mice; wild-type, ^*P = 0.0022 vs. ND, TRPV4-KO, P = 0.1189 vs. ND, Student’s t test, Mann–Whitney test; scale bars, 50 μm). c Aortic permeability under normal and high-fat conditions in wild-type and TRPV4-KO mice. Relative content of Evans blue per gram aorta normalized to control aorta (n = 3 mice; wild-type, ^*P = 0.0132 vs. ND; TRPV4-KO, P = 0.1210 vs. ND, Student’s t test, unpaired two-tailed t test). All data are shown as the mean ± SEM.

Fig. 4. Specific binding sites of TRPV4 and Nox2.

a Statistics of FRET efficiency (TRPV4–Nox2 vs. Nox2-△4, n = 4–6 Petri dishes, ^*P = 0.0004; one-way ANOVA, Dunnett’s multiple comparisons test; otherwise, no significant difference; NC negative control). b FRET results in aorta ECs from ND, HFD, TRPV4 KO-ND, and TRPV4 KO-HFD mice pretreated with AAV-Flt1 vector, AAV-Flt1-Nox2, or AAV-Flt1-Nox2 mut (HFD, n = 8, ^*P < 0.0001 vs. AAV-Flt1 vector; TRPV4-KO HFD, n = 7, ^*P = 0.4607 vs. AAV-Flt1 vector; ND, n = 7, ^*P = 0.0379 vs. AAV-Flt1 vector; TRPV4-KO, n = 6, P = 0.2590 vs. AAV-Flt1 vector. Student’s t test, Mann–Whitney test for ND, otherwise, Student’s t test, unpaired two-tailed t test). c GST pull-down assay showed that TRPV4 physically interacts with Nox2, the mutation of Nox2 destroys the interaction. All data are the mean ± SEM. △AR1-5 and △1-5, mutation at different binding sites of TRPV4 and Nox2; AAV-Flt1, endothelium-targeting adeno-associated virus.

Fig. 5. Decreasing the TRPV4–Nox2 association lowers vascular permeability, while enhancing TRPV4–Nox2 association increases vascular permeability.

a, f, k, p, u Quantification of relative DCF fluorescence in arterial segments isolated from HFD and TRPV4 KO-HFD mice after injection of AAV-Flt1 vector and AAV-Flt1-Nox2 mut (a, f) and from ND and TRPV4 KO-ND mice after injection of AAV-Flt1 vector, AAV-Flt1-Nox2 and AAV-Flt1-Nox2 mut (k, p, u). (n = 6–7 mice, ^*P < 0.05 vs. AAV-Flt1 vector, Student’s t test, unpaired two-tailed t test, otherwise, no significant difference). b, g, l, q, v Representative photomicrographs of rhodamine phalloidin staining in aortic ECs from HFD and TRPV4 KO-HFD mice after injection of AAV-Flt1 vector and AAV-Flt1-Nox2 mut (b, g) and from ND and TRPV4 KO-ND mice after injection of AAV-Flt1 vector, AAV-Flt1-Nox2 and AAV-Flt1-Nox2 mut (l, q, v). Scale bars, 100 μm. c, h, m, r, w Relative mRNA concentrations of VE-cadherin and ICAM-1 in aortic ECs as in (a–u) (n = 4–9 mice; ^*P < 0.05 vs. AAV-Flt1 vector, Student’s t test, paired t test, otherwise, no significant difference). d, i, n, s, x Relative intensity of FITC-dextran showing permeability of aortic ECs isolated from HFD and TRPV4 KO-HFD mice after injection of AAV-Flt1 vector and AAV-Flt1-Nox2 mut (d, i) and from ND and TRPV4 KO-ND mice after injection of AAV-Flt1 vector, AAV-Flt1-Nox2 and AAV-Flt1-Nox2 mut (n, s, x) (n = 6 mice; ^*P < 0.05 vs. AAV-Flt1 vector, Student’s t test, upaired t test, otherwise, no significant difference). e, j, o, t, y Relative content of Evans Blue per gram aorta from HFD and TRPV4 KO-HFD mice after injection of AAV-Flt1 vector and AAV-Flt1-Nox2 mut (e, j) and from ND and TRPV4 KO-ND mice after injection of AAV-Flt1 vector, AAV-Flt1-Nox2 and AAV-Flt1-Nox2 mut (o, t, y) (n = 3–6 mice; ^*P < 0.05 vs. AAV-Flt1 vector, Student’s t test, Paired t test, otherwise, no significant difference). All data are shown as the mean ± SEM. AAV-Flt1-Nox2 mut, Nox2 mutant AAV with a mutated region in the Nox2 and TRPV4 binding domain.

Fig. 6. Decreasing the TRPV4–Nox2 association with M12 improves obesity-induced vascular permeability.

a Immuno-FRET results from HEK-293 cells transfected with Nox2 and TRPV4. Cells were pretreated with DMSO (0.1%), gambogic acid (1 μM), CB-839 (1 μM), L755507 (5 μM), entrectinib (10 nM), bohemine (1 μM), and M12 (10 nM) for 24 h (n = 7 Petri dishes; ^*P < 0.05 vs. wild-type control TRPV4–Nox2, one-way ANOVA, Tukey’s multiple comparisons test). b Structural formula of M12. c Immuno-FRET results in HFD aortic ECs pretreated with or without 10 nM M12 for 24 h (n = 5, ^*P = 0.0025 vs. HFD, Student’s t test, unpaired two-tailed t test). d Representative images and quantification of relative DCF fluorescence in arterial segments isolated from HFD mice pretreated with or without M12 (10 nM) for 24 h (n = 13, ^*P < 0.0001 vs. HFD, Student’s t test, unpaired two-tailed t test, scale bars, 50 μm). e Representative photomicrographs of rhodamine phalloidin staining in aortic ECs from HFD mice pretreated with or without M12 (10 nM) for 24 h (scale bars, 100 μm). f Relative mRNA concentrations of VE-cadherin and ICAM-1 in aortic ECs from HFD mice (VE-cadherin, n = 6 mice; ^*P = 0.0188 vs. HFD; ICAM-1, n = 4; ^*P = 0.0107 vs. HFD, Student’s t test, Paired t test). g FITC-dextran staining assays showing the relative permeability of aortic ECs from HFD mice (n = 3) pretreated with M12 (^*P = 0.0204 vs. HFD, Student’s t test, paired t test). All data are shown as the mean ± SEM.