Soluble Epoxide Hydrolase Inhibition Protected against Angiotensin II-induced Adventitial Remodeling

Figures

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  Figure 1. sEH was involved in adventitial remodeling process. (A) Schematic of animal model. (B) Representative immunoblots and quantitation of sEH protein expression in aortas of Ang II-infused mice. (C) Vascular 11,12-EET and 14,15-EET levels measured by LC/MS. Data were expressed as Mean ± SEM, n = 8 mice for each group. *p < 0.05 vs. Vehicle. Full-length blots/gels are presented in Supplementary Information.

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  Figure 2. TPPU administration attenuated Ang II-induced adventitial remodeling in C57BL/6 J mice. (A) Vessel sections were performed with hematoxylin and eosin staining, sirius red staining, and immunohistochemical staining of α-SMA and PCNA. (B) Quantitative analysis of adventitial thickness. (C) Quantitative analysis of adventitial α-SMA expression. (D) Quantitative analysis of adventitial PCNA expression. (E) Quantitative analysis of adventitial fibrosis. Data were expressed as Mean ± SEM, n = 8 mice for each group. *p < 0.05 vs. Vehicle, #p < 0.05 vs. Ang II.

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  Figure 3. TPPU administration regulated Calcineurin/NFATc3 signaling in adventitial remodeling mice. (A) Representative immunoblots and quantitation of CnA protein expression in aortas. (B) Measurement of CaN phosphatase activity in aortas. (C) Representative immunoblots and quantitation of p-NFATc3/NFATc3 expression in aortas. (D) Representative immunoblots and quantitation of NFATc3 nuclear translocation in aortas. Data were expressed as Mean ± SEM, n = 8 mice for each group. *p < 0.05 vs. Vehicle, #p < 0.05 vs. Ang II, **p < 0.05 vs. Vehicle (Cytoplasm), ##p < 0.05 vs. Ang II (Cytoplasm), &&p < 0.05 vs. Ang II + TPPU (Cytoplasm), $$p < 0.05 vs. Vehicle (Nucleus), ++p < 0.05 vs. Ang II (Nucleus).

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  Figure 4. Effects of TPPU pretreatment on Ang II-induced AFs activation. (A) Immunofluorescent staining of Vimentin and α-SMA in AFs (Red: Cy3, Green: FITC, blue: DAPI). (B) Representative immunoblots and quantitation of α-SMA and Collagen I protein expression in Ang II-stimulated AFs pretreated with or without TPPU. (C) Measurement of cell proliferation by CCK-8 assay in Ang II-stimulated AFs pretreated with or without TPPU. (D) Measurement of cell migration by transwell assay in Ang II-stimulated AFs pretreated with or without TPPU. Data were expressed as mean ± SEM, n = 4 for each group, *p < 0.05 vs. Control, #p < 0.05 vs. Ang II.

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  Figure 5. TPPU inhibited Ang II-activated Ca2+-calcineurin/NFATc3 signaling in AFs. (A) TPPU prevented Ang II-increased intracellular Ca2+ concentration in AFs. (B) Representative immunoblots and quantitation of CnA protein expression in Ang II-stimulated AFs pretreated with or without TPPU. (C) Measurement of CaN phosphatase activity in Ang II-stimulated AFs pretreated with or without TPPU. (D) Representative immunoblots and quantitation of p-NFATc3/NFATc3 expression in Ang II-stimulated AFs pretreated with or without TPPU. (E) Immunofluorescent staining of NFATc3 in Ang II-stimulated AFs pretreated with or without TPPU (Red: Cy3, blue: DAPI). (F) Representative immunoblots and quantitation of NFATc3 nuclear translocation in Ang II-stimulated AFs pretreated with or without TPPU. Data were expressed as mean ± SEM, n = 4 for each group, *p < 0.05 vs. Control, #p < 0.05 vs. Ang II, **p < 0.05 vs. Control (Cytoplasm), ##p < 0.05 vs. Ang II (Cytoplasm), &&p < 0.05 vs. Ang II + TPPU (Cytoplasm), $$p < 0.05 vs. Control (Nucleus), ++p < 0.05 vs. Ang II (Nucleus).

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  Figure 6. Schematic of the potential mechanisms of sEH inhibition on Ang II-induced adventitial remodeling.