Gut Microbiota-Related Evidence Provides New Insights Into the Association Between Activating Transcription Factor 4 and Development of Salt-Induced Hypertension in Mice

Tian-Hao Liu, Wen-Cong Tao, Qiu-Er Liang, Wan-Qing Tu, Ya Xiao, Li-Guo Chen, Tian-Hao Liu, Wen-Cong Tao, Qiu-Er Liang, Wan-Qing Tu, Ya Xiao, Li-Guo Chen

Abstract

Activating transcription factor 4 (ATF4), which regulates genes associated with endoplasmic reticulum stress, apoptosis, autophagy, the gut microbiome, and metabolism, has been implicated in many diseases. However, its mechanistic role in hypertension remains unclear. In the present study, we investigated its role in salt-sensitive hypertensive mice. Wild-type (WT) C57BL/6J mice were used to establish Atf4 knockout (KO) and overexpression mice using CRISPR-Cas9 and lentiviral overexpression vectors. Then, fecal microbiota transplantation (FMT) from Atf4 ± mice and vitamin K2 (VK2) supplementation were separately carried out in high-salt-diet (8% NaCl)-induced mice for 4 weeks. We found that Atf4 KO inhibited and Atf4 overexpression enhanced the increase in blood pressure and endothelial dysfunction induced by high salt intake in mice, while regulating the gut microbiota composition and VK2 expression. It was further verified that ATF4 is involved in the regulation of salt-sensitive hypertension and vascular endothelial function, which is achieved through association with gut microbiota and may be related to VK2 and different bacteria such as Dubosiella. In addition, we found that VK2 supplementation prevents the development of salt-sensitive hypertension and maintains vascular endothelial function; moreover, VK2 supplementation increases the abundance of intestinal Dubosiella and downregulates the relative expression of Atf4 in the thoracic aorta of mice. We conclude that ATF4 plays an important role in regulating gut microbiota and VK2 production, providing new insights into the association between ATF4 and development of salt-induced hypertension in mice, meanwhile contributing to the development for a new preventive strategy of hypertension.

Keywords: gene regulation; gut microbiota; hypertension; mice; vitamin K2.

Copyright © 2020 Liu, Tao, Liang, Tu, Xiao and Chen.

Figures

FIGURE 1
FIGURE 1
The cartoon of experimental design to investigate the potential mechanism of ATF4 participating in high-salt diet-induced hypertension. Wild-type (WT) C57BL/6J mice, Atf4 knockout (Atf4±) and Atf4 overexpression mice were used and high-salt-diet (8% NaCl) for 4 weeks was used to induce hypertensive mice. Then, fecal microbiota transplantation from Atf4± mice and vitamin K2 supplementation were separately carried out to verify the gut microbiota-related evidence of the association between ATF4 and blood pressure. ATF4, activating transcription factor 4; KO, Atf4± mice; WT, wild-type mice; NO, nitric oxide; VEGF, vascular endothelial growth factor; ANGI, angiotensin I; ET-1, endothelin-1; KO-ND, Atf4± mice with natural diet; KO-HSD, Atf4± mice with high-salt diet; ND, WT mice with natural diet; HSD, WT mice with high-salt diet; OE, Atf4 overexpression with high-salt diet; OC, Atf4 overexpression control with high-salt diet; FMT, fecal microbiota transplantation; VK2, vitamin K2.
FIGURE 2
FIGURE 2
ATF4 altered SBP and endothelial function-related factors in high-salt-induced hypertensive mice. (A) ATF4 altered SBP in high-salt-induced hypertensive mice. ND vs HSD, HSD vs OE, OE vs OC, *p < 0.05 and **p < 0.01. (B) ATF4 altered the expression of NO in high-salt-induced hypertensive mice. (C) ATF4 altered the expression of VEGF in high-salt-induced hypertensive mice. (D) ATF4 altered the expression of ANGI in high-salt-induced hypertensive mice. (E) ATF4 altered the expression of ET-1 in high-salt-induced hypertensive mice. Data are presented as mean ± SD; ns, no significance, *p < 0.05 and **p < 0.01, n = 6–8; statistical comparisons were performed using Student t-test or one-way analysis of variance (ANOVA). ATF4, activating transcription factor 4; KO, Atf4± mice; WT, wild-type mice; SBP, systolic blood pressure; NO, nitric oxide; VEGF, vascular endothelial growth factor; ANGI, angiotensin I; ET-1, endothelin-1; KO-ND, Atf4± mice with natural diet; KO-HSD, Atf4± mice with high-salt diet; ND, WT mice with natural diet; HSD, WT mice with high-salt diet; OE, Atf4 overexpression with high-salt diet; OC, Atf4 overexpression control with high-salt diet.
FIGURE 3
FIGURE 3
ATF4 altered the composition of gut microbiota and expression levels of the microbial metabolite VK2 in high-salt-induced hypertensive mice. (A) Relative abundance plots displaying the differences in the microbial community structure at the phylum level. (B) Relative abundance plots displaying the differences in the general microbial community structure. (C) PCA plots were used to visualize differences in weighted UniFrac distances of samples of OTUs from different groups (R2 = 0.3264, p = 0.001). (D) ATF4 altered the expression of VK2. Data are presented as mean ± SD; **p < 0.01, n = 6–8; statistical comparisons were performed using Student t test or one-way analysis of variance (ANOVA). ATF4, activating transcription factor 4; KO, Atf4± mice; WT, wild-type mice; SBP, systolic blood pressure; NO, nitric oxide; VEGF, vascular endothelial growth factor; ANGI, angiotensin I; ET-1, endothelin-1; KO-ND, Atf4± mice with natural diet; KO-HSD, Atf4± mice with high-salt diet; ND, WT mice with natural diet; HSD, WT mice with high-salt diet; OE, Atf4 overexpression with high-salt diet; OC, Atf4 overexpression control with high-salt diet.
FIGURE 4
FIGURE 4
Microbial correlation in high-salt-induced hypertensive mice. (A) Spearman correlation analysis between top 20 genera and environmental factors. Red represents positive correlation, whereas blue represents negative correlation; the darker the color, the stronger the correlation. ∗0.01 < p ≤ 0.05, ∗∗0.001 < p ≤ 0.01, ∗∗∗p ≤ 0.001. (B) RDA analysis of environmental factors. The angle between the arrows of different environmental factors represents positive and negative correlations (acute angle: positive correlation; obtuse angle: negative correlation; right angle: no correlation). ATF4, activating transcription factor 4; KO, Atf4± mice; WT, wild-type mice; SBP, systolic blood pressure; NO, nitric oxide; VEGF, vascular endothelial growth factor; ANGI, angiotensin I; ET-1, endothelin-1; KO-ND, Atf4± mice with natural diet; KO-HSD, Atf4± mice with high-salt diet; ND, WT mice with natural diet; HSD, WT mice with high-salt diet; OE, Atf4 overexpression with high-salt diet; OC, Atf4 overexpression control with high-salt diet.
FIGURE 5
FIGURE 5
FMT using Atf4± mice fecal microbiota improved SBP and altered expression of endothelial function-related factors in high-salt-induced hypertensive mice. (A) FMT maintained SBP. (B) FMT upregulated the expression of NO. (C) FMT downregulated the expression of VEGF. (D) FMT downregulated the expression of ANGI. (E) FMT downregulated the expression of ET-1. (F) Examination of endothelial morphology by transmission electronic microscope (TEM). IEL, internal elastic lamina; N, nucleus; M, mitochondrion; RER, rough endoplasmic reticulum; TJ, tight junction; AP, autophagy. Data are presented as mean ± SD; **p < 0.01, n = 6–8; statistical comparisons were performed using Student t-test or one-way analysis of variance (ANOVA). FMT, fecal microbiota transplantation; SBP, systolic blood pressure; NO, nitric oxide; VEGF, vascular endothelial growth factor; ANGI, angiotensin I; ET-1, endothelin-1; ND, WT mice with natural diet; HSD, WT mice with high-salt diet.
FIGURE 6
FIGURE 6
FMT using Atf4± mice fecal microbiota altered the composition of gut microbiota and expression of the microbial metabolite VK2 in high-salt-induced hypertensive mice. (A) Venn diagram of OTUs. Different colors represent different groups. (B) Relative abundance plots displaying the differences in the microbial community structure at the phylum level. (C) Relative abundance plots displaying the differences in the general microbial community structure. (D) NMDS plots were used to visualize differences in weighted UniFrac distances at the genus level of samples from different groups. (E) The 11 taxonomic groups associated with FMT. *p < 0.05 and **p < 0.01. (F) FMT upregulated the expression of VK2. Data are presented as mean ± SD; **p < 0.01, n = 6–8; statistical comparisons were performed using Student t-test or one-way analysis of variance (ANOVA). FMT, fecal microbiota transplantation; VK2, vitamin K2; KO-ND, Atf4± mice with natural diet; ND, WT mice with natural diet; HSD, WT mice with high-salt diet.
FIGURE 7
FIGURE 7
VK2 maintained SBP and altered endothelial function-related factors in high-salt-induced hypertensive mice. (A) VK2 altered SBP. (B) VK2 upregulated the expression of NO. (C) VK2 downregulated the expression of VEGF. (D) VK2 downregulated the expression of ANGI. (E) VK2 downregulated the expression of ET-1. Data are presented as mean ± SD; *p < 0.05 and **p < 0.01, n = 6–8; statistical comparisons were performed using Student t-test or one-way analysis of variance (ANOVA). SBP, systolic blood pressure; VK2, vitamin K2; NO, nitric oxide; VEGF, vascular endothelial growth factor; ANGI, angiotensin I; ET-1, endothelin-1; ND, WT mice with natural diet; HSD, WT mice with high-salt diet.

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