A novel TRPV4-specific agonist inhibits monocyte adhesion and atherosclerosis

Abstract

TRPV4 ion channel mediates vascular mechanosensitivity and vasodilation. Here, we sought to explore whether non-mechanical activation of TRPV4 could limit vascular inflammation and atherosclerosis. We found that GSK1016790A, a potent and specific small-molecule agonist of TRPV4, induces the phosphorylation and activation of eNOS partially through the AMPK pathway. Moreover, GSK1016790A inhibited TNF-α-induced monocyte adhesion to human endothelial cells. Mice given GSK1016790A showed increased phosphorylation of eNOS and AMPK in the aorta and decreased leukocyte adhesion to TNF-α-inflamed endothelium. Importantly, oral administration of GSK1016790A reduced atherosclerotic plaque formation in ApoE deficient mice fed a Western-type diet. Together, the present study suggests that pharmacological activation of TRPV4 may serve as a potential therapeutic approach to treat atherosclerosis.

Keywords: AMPK; GSK1016790A; TRPV4; atherosclerosis; shear stress.

Figure 1. GSK1016790A activates eNOS signaling in human endothelial cells

A.-D., Time course (A-B) and dose-dependency (C-D) of GSK1016790A (GSK101) stimulation in HUVECs. HUVECs were treated with 10 nM GSK101 for indicated times or with indicated concentration of GSK101 for 15 min, then phosphorylation of eNOS, Akt, and AMPK were analyzed by Western blotting. Data were expressed as means ± SEM. E., HCAECs were treated with vehicle or 10 nM GSK101 for 15 min, then phosphorylation of eNOS, Akt, and AMPK were analyzed by Western blotting.

Figure 2. GSK1016790A-mediated signaling is calcium- and TRPV4-dependent

A. HUVECs were pretreated with vehicle (0.1% DMSO), or calcium chelator BAPTA (3 μM) plus EGTA (500 μM) for 30 min, and then exposed to GSK1016790A (GSK101, 10 nM) for 15 min. B. HUVECs were pretreated with vehicle (H₂O), or non-selective TRPV4 inhibitor Ruthenium Red (RuR; 5 μM) for 30 min, and then exposed to GSK101 (10 nM) for 15 min. C. HUVECs were pretreated with vehicle (0.1% DMSO), or TRPV4 specific antagonist GSK2193874 (GSK874, 100 nM) for 30 min, and then exposed to GSK101 (10 nM) for 15 min. D. HUVECs were transfected with 25 nM non-target control siRNA (siNC) or siRNA against TRPV4 (siTRPV4) for 48 h and then exposed to GSK101 (10 nM) for 15 min. Cell lysates were analyzed for Western blots using antibodies indicated.

Figure 3. GSK1016790A activates eNOS partially through the CaMKK/AMPK pathway

A. HUVECs were pretreated with vehicle (0.1% DMSO), or the CaMMK inhibitor STO-609 (1 μM) for 30 min, and then exposed to GSK1016790A (GSK101, 10 nM) for 15 min. B. HUVECs were pretreated with vehicle (0.1%DMSO), or the AMPK inhibitor Compound C (3 μM) for 30 min, and then exposed to GSK101 (10 nM) for 15 min. C. Aortas from C57BL/6J mice were pretreated with vehicle (0.1%DMSO), or STO-609 (1 μM), or Compound C (3 μM) for 30 min, then GSK101 (10 nM)-induced vasorelaxation of phenylephrine (PE, 1 μM)-precontracted aorta were determined. Values are mean ± SEM, ***p < 0.001 vs. vehicle control group. ^###p < 0.001 vs. GSK101 group.

Figure 4. GSK1016790A treatment induces eNOS and AMPK phosphorylation in mouse aorta

A.-B. Three-month old male C57BL/6J mice were injected (i.p.) with GSK1016790A (GSK101, 50 μg/kg body weight) or vehicle (n = 17 for detecting p-eNOS Ser1177; n = 6 for detecting p-AMPK and p-ACC). Thirty minutes after the treatment, mice were sacrificed and aortic tissues were harvested. Tissue lysates were analyzed by Western blots. C.-D. En face immunostaining of mouse aortic endothelium showing that GSK101 increased the phosphorylation of eNOS (p-eNOS Ser1177, red) and AMPK phosphorylation (p-AMPK Thr172, red) in aortic endothelium, and VE-cadherin (green) was used to label endothelial cells, DAPI was used to counterstain cell nuclei, bar = 30 μm.

Figure 5. GSK1016790A attenuates monocyte adhesion to endothelial cells in vitro and in vivo

A. HUVECs were pretreated with vehicle (DMSO), or GSK1016790A (GSK101, 1 nM and 10 nM) for 2 h, and then exposed to TNF-α (10 ng/ml) or vehicle (PBS) for an additional 6 h. Then, U937 monocytes was added for 30 min. Images were taken from representative optical fields showing endothelial cells (cobblestone shape) and adhering U937 monocytes (small, round cells) in the co-culture. B. Summary data for adherent monocytes described in A. C. and D. HUVECs were treated as described in A, then mRNA and protein expression of ICAM1 and VCAM1 were determined by qPCR and Western blotting, respectively. Values are mean ± SEM, *p < 0.05, ***p < 0.001 vs. vehicle control group. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. TNF-α group. E., HUVECs were pretreated with 100 μM eNOS inhibitor L-NAME for 30 min before treatment with GSK101 described in A. The images of U937 monocytic cells adherent to ECs were presented. F. Summary data for panel E. Values are mean ± SEM, ***p < 0.001 vs. TNF-α group. ^##p < 0.01 vs. TNF-α +GSK101 group. G. Male C57BL/6 mice were pretreated with vehicle or GSK1016790A (10 mg/kg/d, by oral gavage) for 3 days, followed by vehicle or TNF-α injection (i.p.) for 4 h, intravital microscopy analysis was then performed to evaluate leukocyte adhesion to endothelium in the mesenteric microcirculation. Representative still images were shown for mice treated with vehicle (PBS), GSK101, TNF-α, or TNF-α plus GSK101. Summary data are provided in H. Values are mean ± SEM; n = 6-10; ***p < 0.001 versus PBS group, ^###p < 0.001 versus TNF-α group.

Figure 6. GSK1016790A attenuates the development of atherosclerotic lesions in ApoE−/− mice

A. Representative images of atherosclerotic lesion formation in aortic arch of vehicle and GSK1016790A (GSK101)-treated ApoE^−/−mice. B. Frozen sections from the aortic sinus of ApoE^−/− mice treated with vehicle or GSK101 for 4 weeks were stained with Oil Red O. The area of the Oil Red O-positive atherosclerotic lesions was quantified in C. Each symbol in the scatter plots represents the lesion area of one individual mouse. Values are mean ± SEM. D. Representative photographs from en face analysis of aortas from ApoE^−/− mice treated with vehicle or GSK101 for 4 weeks. The pictures presented were a composite of 4-6 images captured at different regions of the same aorta. The percentage of surface area of the Oil Red O-positive atherosclerotic lesions from the en face preparation relative to total luminal surface area of aorta was quantified in E. Each symbol in the scatter plots represents the lesion area percentage of one individual mouse. Values are mean ± SEM; ***p < 0.001 vs. vehicle group. F. GSK101 decreased macrophage content in atherosclerotic plaques. Aortic sinus from mice treated with vehicle or GSK101 were stained with rat anti-CD68 antibody to display macrophage content (Red), DAPI (Blue) was used to counterstain the nuclei, bar = 30 μm. L, lumen; A, atheroma; M, media. G. Quantification of CD68 positive staining of macrophages in panel F.