Treatment of hypertension by increasing impaired endothelial TRPV4‐KCa2.3 interaction

Figure 1. Impairment of the physical and functional interaction of TRPV4‐KCa2.3 channels in hypertension AThe hypothesis investigated in this study was that endothelial TRPV4‐KCa2.3 interaction modulates blood pressure, and impairment of the interaction is related to hypertension. So, strengthening the interaction with a small‐molecule drug, JNc‐440, would show antihypertensive effects.BImmuno‐FRET experiments in arterial sections from normotensive and hypertensive humans. Left: representative figures for FRET. White dotted line, autofluorescence of elastin underneath the endothelium; right: analysis of 21 normotensive and 15 hypertensive individuals (# P < 0.0001, unpaired t‐test vs. control). Scale bar, 20 μm.C–EImmuno‐FRET experiments in arterial sections from (C) mice on a high‐salt diet (8% NaCl), (D) mice with L‐NNA intake (0.5 g/l in the drinking water), or (E) mice with AngII delivery (infused at 500 ng/kg/min) (# P < 0.0001, unpaired t‐test vs. control).FFluorescent K+ efflux results showing the TRPV4 agonist GSK1016790A (GSK)‐induced K+ efflux in primary cultured ECs from normal mice and mice on a high‐salt diet (left). Six mice were tested for each treatment and analyzed…

Figure 2. Increasing the TRPV4‐KCa2.3 interaction lowers blood pressure, but dissociation of TRPV4‐KCa2.3 interaction increases blood pressure A–HMice were intravenously injected with AAV‐Flt1‐KCa2.3 and the AAV‐Flt1 vector served as a control. (A) Representative en face (left) and cross‐sectional (right) fluorescence images of AAV‐Flt1‐KCa2.3 or vector (flag‐tag) expression in AAV‐Flt1‐infected mice. Green, autofluorescence of elastin underneath the endothelium (excitation, 488 nm); red, anti‐flag. Scale bars, 10 μm. (B) Seven mice were tested for the effects of AAV‐Flt1‐KCa2.3 or vector in immuno‐FRET experiments (# P < 0.0001 vs. control, unpaired t‐test). (C, F) K+ efflux from primary cultured ECs (# P < 0.0001 vs. control, unpaired t‐test). (D, G) Vasodilation assays by wire myography (GSK1016790A (GSK) and ACh in freshly isolated arterial segments) (*P = 0.047, # P < 0.0001 vs. control, two‐way ANOVA). (E, H) Time course of the changes in mean arterial pressure (∆MAP) in wild‐type (C–E) and TRPV4‐knockout (KO) (F–H) mice after tail injection of AAV‐Flt1‐KCa2.3, AAV‐Flt1 vector, or vehicle (injection solution).I–OMice were intravenously injected with AAV‐Flt1‐KCa2.3‐17C; the AAV‐Flt1…

Figure 3. Increasing the TRPV4‐KCa2.3 interaction with JNc‐440 is antihypertensive ALeft: structural formula of JNc‐440. Right: affinity of JNc‐440 for both TRPV4 and KCa2.3. Biotinylated JNc‐440 (JNc‐440‐biotin) pulled down TRPV4 or KCa2.3 in primary cultured ECs.B–G(B) Immuno‐FRET experiments in arterial sections, (C) K+ efflux from primary cultured ECs, (D) vasodilation assays by wire myography (GSK and ACh) in freshly isolated arterial segments, and (E) mean arterial pressure. (F) Effect of JNc‐440 on blood pressure in normotensive mice. (G) Effect of JNc‐440 (1 mg/kg; daily for 1 week) on blood pressure in TRPV4‐knockout (KO) mice treated with a high‐salt diet, L‐NNA, or AngII. WT mice were treated with a high‐salt diet, L‐NNA, or AngII and then intravenously injected with JNc‐440 (1 mg/kg) or vehicle for 2 h (B–F) or daily for 1 week (G). Data are mean ± SD. (B, C) # P < 0.0001 vs. control, unpaired t‐test. (D) *P = 0.025 (high‐salt diet), *P = 0.008 (L‐NNA treatment), or *P = 0.021 (AngII treatment), # P < 0.0001 vs. control, two‐way ANOVA. (E) *P = 0.001, # P < 0.0001 vs. control, two‐way ANOVA. Source data are available online for this figure.