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. 2020 Aug 17;130(9):4999–5010. doi: 10.1172/JCI136621

Figure 5. Abrogation of the 12(S)-HpETE/TRPV1 interaction protects against diabetes-induced endothelial dysfunction.

Figure 5

(A) Schematic of TRPV1 corresponding to amino acids 701–711 within the TRP box at the C-terminus of human TRPV1 linked to a TAT linker protein for intracellular entry. (B) Patch clamp experiments show that 700 nM 12(S)-HpETE evokes currents in endothelial cells loaded with 1 μM TAT linker, but not 1 μM V1-cal. Representative patch results from n = 10 independent experiments. (C) 12(S)-HpETE–induced mitochondrial calcium changes detected by flow cytometry [time point of addition of 12(S)-HpETE is indicated by the arrow] in human endothelial cells preincubated with either 1 μM V1-cal or 1 μM of a scrambled version of V1-cal (V1-scr) or TAT linker protein serving as controls. ***P < 0.001 vs. corresponding time point in V1-cal or EtOH, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. TAT or V1-scr, 2-way ANOVA/Bonferroni, n = 5 independent experiments. (D) Assessment of mitochondrial function to evaluate effects of 1 μM V1-cal versus 1 μM TAT linker only (top 2 panels) or 1 μM V1-scr (bottom panel) on mitochondrial OCR induced by 1 μM 12(S)-HpETE. After 3 measurements, oligomycin was added before FCCP used to induce maximum respiration. *P < 0.05, **P < 0.01, 2-way ANOVA/Bonferroni. Variants were analyzed in triplicate on Seahorse miniplates, and n = 5 independent experiments were performed. All graphs show mean ± SEM. (E) Cch-induced vasorelaxation shows that repetitive intravenous application on 4 consecutive days of the V1-cal peptide (1 mg/kg/d), but not of the V1-scr peptide or the TAT linker protein only, to diabetic WT mice reduced endothelial dysfunction induced by diabetes (STZ). Sodium citrate (NaCi) was used as vehicle control. ***P < 0.001 vs. as indicated, 2-way ANOVA/Bonferroni. Means were generated for each individual animal by analysis of 2 arteries; n = 5 animals per group. Graph shows mean ± SEM.