ARTICLE
Received 19 Nov 2015 | Accepted 13 Jul 2016 | Published 2 Sep 2016
DOI: 10.1038/ncomms12565 OPEN
Vascular microRNA-204 is remotely governed by the microbiome and impairs endothelium-dependent vasorelaxation by downregulating Sirtuin1
Ajit Vikram1,2,3,4, Young-Rae Kim1,2,3,4, Santosh Kumar1,2,3,4, Qiuxia Li1,2,3,4, Modar Kassan1,2,3,4, Julia S. Jacobs1,2,3,4 & Kaikobad Irani1,2,3,4,5
Gut microbiota promotes atherosclerosis, and vascular endothelial dysfunction, signalled by impaired endothelium-dependent vasorelaxation, is an early marker of atherosclerosis. Here we show that vascular microRNA-204 (miR-204) expression is remotely regulated by the microbiome, and impairs endothelial function by targeting the Sirtuin1 lysine deacetylase (Sirt1). MiR-204 is downregulated, while Sirt1 is upregulated, in aortas of germ-free mice. Suppression of gut microbiome with broad-spectrum antibiotics decreases miR-204, increases Sirt1 and bioavailable vascular nitric oxide, and improves endothelium-dependent vasorelaxation in mouse aortas. Antibiotics curtail aortic miR-204 upregulation, and rescue decline of aortic Sirt1 and endothelium-dependent vasorelaxation, triggered by high-fat diet feeding. Improvement of endothelium-dependent vasorelaxation by antibiotics is lost in mice lacking endothelial Sirt1. Systemic antagonism of miR-204 rescues impaired endothelium-dependent vasorelaxation and vascular Sirt1, and decreases vascular inammation induced by high-fat diet. These ndings reveal a gut microbe-vascular microRNASirtuin1 nexus that leads to endothelial dysfunction.
1 Cardiovascular Division, Department of Medicine, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242, USA. 2 Abboud Cardiovascular Research Center, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242, USA. 3 Fraternal Order of Eagles Diabetes Research Center, University of Iowa, Iowa City, Iowa 52242, USA. 4 Pappajohn Biomedical Institute, University of Iowa, Iowa City, Iowa 52242, USA. 5 Heart and Vascular Center, University of Iowa Hospitals and Clinics, Iowa City, Iowa 52242, USA. Correspondence and requests for materials should be addressed to A.V.(email: mailto:[email protected]
Web End [email protected] ) or to K.I. (email: mailto:[email protected]
Web End [email protected] ).
NATURE COMMUNICATIONS | 7:12565 | DOI: 10.1038/ncomms12565 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications 1
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12565
The gut ora, consisting of trillions of microbes, plays an essential role in dietary energy harvest. The gut microbiome has been implicated in the pathogenesis
of metabolic disorders such as diabetes1 and obesity2. The microbiome can also contribute to the development of atherosclerotic vascular disease36. Metabolites of dietary phospholipids and nutrients produced by gut ora and found in the systemic circulation contribute to vascular inammation and atherosclerosis3,5. On the other hand, the microbiome also participates in retarding atherosclerosis. Gut bacteria generate metabolites of a class of dietary avonoids that decrease atherosclerotic burden by stimulating reverse cholesterol transport in macrophages7.
Endothelial dysfunction, manifested as impaired endothelium-dependent vasorelaxation and vascular inammation, is a precursor and strong predictor of atherosclerosis8. Sirtuin1 (Sirt1) is a class III histone deacetylase expressed in cell types comprising the vascular wall that stimulates endothelial nitric oxide (NO) by deacetylating endothelial nitric oxide synthase (eNOS), promotes endothelium-dependent vasorelaxation, and protects against endothelial dysfunction and atherosclerosis9,10. Sirt1 expression is governed by microRNAs11. For example, miR-217 and miR-212, both of which directly target Sirt1, promote endothelial cell senescence and suppress in vitro angiogenesis12,13. We hypothesized and prove that the expression of vascular Sirt1 is an important determinant of gut microbiome-dependent endothelial function, and vascular miR-204, one of the microRNAs whose expression is remotely controlled by the microbiome, targets endothelial Sirt1 leading to endothelial dysfunction.
ResultsMicrobiome stimulates the expression of vascular miR-204. Initially, we sought to identify the effect of the microbiota on vascular microRNA expression proles. Unbiased microRNA nCounter expression arrays were performed and compared in aortas of microbiota-free germ-free mice (GFM) and control pathogen-free mice (PFM). Of the 578 microRNAs assayed, 15 were found to be signicantly downregulated, while 5 were upregulated in aortas of GFM compared with PFM (Fig. 1a; Supplementary Fig. 1a). Global microRNA expression, and expression of the most abundant vascular microRNAs, did not differ between GFM and PFM (Supplementary Fig. 1b,c). Similarly, aortic expression of microRNA processing enzymes Dicer and Drosha were not altered in germ-free conditions (Supplementary Fig. 1d). Among the microRNAs downregulated by nCounter array in GFM aortas (Supplementary Fig. 2d), miR-204 and miR-148a were conrmed by quantitative PCR (qPCR), with a strong trend for miR-9 (Supplementary Fig. 2e). Since miR-204 was the most robustly downregulated microRNA, we henceforth focused our attention on it. Decrease of miR-204 in GFM aortas (Fig. 1b) was validated using different internal controls (Supplementary Fig. 2c) and expression of miR-204 precursors in the aorta followed a trend similar to that of the mature microRNA (Fig. 1b). In situ hybridization showed that miR-204 is expressed in both the endothelium and tunica media of mouse aortas (Supplementary Fig. 3).
Sirt1 is targeted by miR-204 and is upregulated in GFM. Computational microRNAmRNA algorithms predicted Sirt1 to be in the top 100 genes (0.5 percentile) targeted by the conserved microRNAs downregulated by nCounter array in aortas of GFM (Fig. 1c). Among these microRNAs, miR-204, miR-9, miR-142-3p and miR-199a-5p have been experimentally shown to down-regulate Sirt1 (refs 1416). Focusing on miR-204, we conrmed
that it targets Sirt1 in vascular endothelial cells using miR-204 modulators (mimic, inhibitor) and Sirt1 30-untranslated region (UTR) reporter assay (Fig. 1d,e; Supplementary Fig. 4). Furthermore, Sirt1 was upregulated in several tissues, including the aortas (Fig. 1f), skeletal muscle and lungs, but not in the heart and liver of GFM (Supplementary Fig. 2f). Because miR-204 is in intron 6 of the Trpm3 gene located on chromosome 9q21.12, and is co-expressed with Trpm3 (ref. 17), we also measured Trpm3 expression. Paralleling miR-204 expression, Trpm3 was downregulated in GFM aortas (Fig. 1g).
Antibiotics decrease miR-204 and increase vascular Sirt1. We next asked whether suppressing gut ora biomass with broad-spectrum oral antibiotics results in changes in vascular miR-204 and Sirt1 expression similar to that observed in GFM. Wild-type C57Bl/6 mice isolated from the colony, housed in separate cages and treated with antibiotics in drinking water for 6 weeks were slightly leaner than their untreated counterparts but had similar plasma lipids (Supplementary Fig. 5a,b). Mice on antibiotics had markedly lower faecal microbial load (Supplementary Fig. 6). Administration of antibiotics decreased aortic miR-204 and Trpm3 (Fig. 2ac), while increasing aortic Sirt1 (Fig. 2dg). Discontinuation of antibiotics and co-housing with mice not on antibiotics reversed changes in aortic expression of miR-204, Trpm3 and Sirt1 (Fig. 2ag). Immunohistochemistry showed that total aortic Sirt1 as well as endothelial Sirt1 was upregulated in mice receiving antibiotics (Fig. 2dg).
Antibiotics promote endothelial function via Sirt1. Because of the important role of Sirt1 in eNOS signalling, we questioned whether the gut microbiome stimulates vascular NO and endothelium-dependent vasorelaxation. Antibiotic administration to mice upregulated eNOS (Fig. 2d,e), and improved endothelium-dependent vasorelaxation (Fig. 2h), in mouse aortas. Repopulation of the microbiome by discontinuation of antibiotics and co-habitation with mice not on antibiotics reversed changes in aortic expression of eNOS and decreased endothelium-dependent vasorelaxation (Fig. 2d,e,h). To determine the role of endothelial Sirt1 in the microbiota-vascular axis, we examined the effect of antibiotics on endothelium-dependent vasorelaxation in mice with conditional deletion of endothelial Sirt1 (eSirt1 / ). In contrast to control wild-type, Sirt1/ and
VE-Cad-Cre mice, antibiotics paradoxically impaired endothelium-dependent vasorelaxation in eSirt1 / mice (Fig. 2hk), showing an obligatory role of endothelial Sirt1 in improved vasorelaxation achieved by suppressing the microbiome.
Sirt1 rescues miR-204-induced endothelial dysfunction. To establish a causal role for miR-204-induced downregulation of Sirt1 in endothelial dysfunction, miR-204 mimic was transfected into mouse aortas ex vivo resulting in robust upregulation of aortic miR-204 (Supplementary Fig. 7a). MiR-204 mimic suppressed endothelial Sirt1 (Supplementary Fig. 7b) and decreased endothelium-dependent vasorelaxation (Fig. 2l). This decrease in endothelium-dependent vasorelaxation was rescued by adenoviral-mediated reconstitution of endothelial Sirt1 (lacking the 30-UTR) in mouse aortas (Fig. 2l; Supplementary
Fig. 7b), indicating that miR-204 mimic impairs endothelial function, at least in part, by suppressing Sirt1 expression.
Diet-microbiome interaction governs miR-204 and Sirt1. We then questioned whether diet-induced endothelial dysfunction is also dependent on the microbiome and triggered by miR-204-mediated downregulation of vascular Sirt1. To address this, we rst measured aortic expression of Sirt1 and miR-204 in a high-fat
2 NATURE COMMUNICATIONS | 7:12565 | DOI: 10.1038/ncomms12565 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12565 ARTICLE
a
b
c
d
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
miR-204
PFM GFM
Luciferase activity
120
miR-204
1.2
6
microRNAs targeting mRNA
Sirt1
80
0.8
*
log 10 Pvalue (GFM versus PFM)
* *
4
40
0.4
2
Sirt2-7
0
0
0.0
25
50
100
SC miR-
204-M
mature miR-204
Pri-and Pre-miR-204
mRNAs (% total)
e
f
g
miR-204-M
0 72
24
SC
180
120
*
120
80
Sirt1
0 72
(h) 24
Sirt1
Trpm3
0.60
0.45
0.30
0.15
0.00
0.15
0.30
0.45
0.60
60
40
*
110 kDa
38 kDa
0
0
log2 fold change (GFM versus PFM)
Gapdh
PFM
GFM
PFM
GFM
Figure 1 | MiR-204 is downregulated and Sirt1 is upregulated in aortas of GFM. (a) Volcano plot of differentially expressed microRNAs in aortas of germ-free mice (GFM). Difference in the expression of aortic microRNAs between GFM and pathogen-free mice (PFM) is plotted on the x axis (log2 scale), and signicance is plotted on the y axis (log10 scale). Upregulated or downregulated microRNAs are indicated in red and blue, respectively. n 3 mice for each
microRNA. (b) Decreased mature and precursor miR-204 expression in aortas of GFM by qPCR (shown as % of PFM). n 4 for each group, *Po0.05 versus
PFM. (c) In silico analysis of mRNAs targeted by microRNAs that were downregulated by nCounter assay in aortas of GFM. Number of downregulated microRNAs targeting a specic mRNA is shown on the y axis and % of total mRNA is shown on the x axis. MicroRNA-SVR score is colour-coded with thresholdo 0.50 in grey, o 0.75 in blue ando 0.90 in red. Lower SVR score indicates stronger predicted microRNAmRNA interaction. Sirt1 is targeted
by microRNAs downregulated in GFM aortas, and based on the number of microRNAs targeting a specic gene it was predicted to be in top 0.5% of putatively regulated genes. (d) MiR-204 mimic (miR-204-M) decreases Sirt1 30-UTR luciferase reporter activity. SC, scrambled control microRNA.
*Po0.05 versus SC, n 5 independent experiments. (e) MiR-204-M (20 nM) decreases Sirt1 expression in human umbilical vein endothelial cells (HUVECs)
in a time-dependent manner. Representative of three independent experiments is shown. (f,g) Increased Sirt1 (f) and decreased Trpm3 (g) mRNA expression in aortas of GFM (shown as % of PFM). n 4 for each group, *Po0.05 versus PFM. Independent sample t-test was used. Data shown as mean and error bar
represents s.e.m.
diet (HFD) feeding model of endothelial dysfunction. HFD feeding of C57Bl/6 mice for 8 weeks upregulated aortic miR-204 and its precursors (Fig. 3ac), and downregulated total aortic and endothelial Sirt1 (Fig. 3df). Antibiotics administered during the last 6 weeks of the HFD feeding period decreased aortic miR-204 to basal levels (Fig. 3ac), and restored aortic and endothelial Sirt1 (Fig. 3df). Discontinuation of antibiotics and co-habitation of the mice with littermates not on antibiotics for 2 weeks reversed changes in aortic miR-204 (Fig. 3ac) and Sirt1 (Fig. 3df).
Antibiotics reverse HFD-induced endothelial dysfunction via Sirt1. HFD feeding impaired endothelium-dependent vasorelaxation (Fig. 3h), and reduced bioavailable vascular NO, in mouse aortas (Fig. 3i). Broad-spectrum antibiotics rescued this decline in endothelium-dependent vasorelaxation (Fig. 3h) and bioavailable NO (Fig. 3i). Cessation of antibiotics and re-housing the mice with those not on antibiotics negated improvement in endothelium-dependent vasorelaxation and bioavailable NO (Fig. 3h,i). Importantly, rescue of endothelium-dependent vasorelaxation by antibiotics was lost in eSirt1 / mice (Fig. 3j), underscoring the role of endothelial Sirt1 in mediating the benecial effect of gut microbiota suppression on vascular endothelial function.
Antibiotics promote endothelial Stat3 signalling. We next sought to understand the molecular mechanism responsible for the regulation of vascular miR-204 expression by the gut microbiome. The transcription factor Stat3 represses miR-204 expression in vascular smooth muscle cells and pancreatic beta cells18,19. We therefore explored the role of Stat3 signalling in regulation of vascular miR-204 by the gut microbiome. Inhibition of Stat3 signalling in endothelial cells with a dominant-negative Stat3 construct stimulated miR-204 expression (Supplementary
Fig. 8a). Moreover, activated (phosphorylated) Stat3 was diminished in aortas of mice on HFD (Fig. 3f; Supplementary Fig. 8b,c). Suppression of the microbiome with antibiotics rescued aortic Stat3 signalling, while reconstitution of the gut ora diminished phospho-Stat3 (Fig. 3f; Supplementary Fig. 8b,c). Importantly, phosphorylation of Stat3, and changes in its phosphorylation with HFD and antibiotics, was seen principally in the endothelium (Supplementary Fig. 8b,c), indicating that endothelial Stat3 signalling is an important target of the gut microbiome.
MiR-204 mediates HFD-induced endothelial dysfunction. We then asked whether vascular miR-204 plays a causative role in endothelial dysfunction and vascular inammation secondary to HFD feeding. Locked nucleic acid anti-miR-204 was systemically delivered with osmotic mini-pumps for the nal 6 weeks of the 8-week HFD feeding period, resulting in robust suppression of miR-204 in mouse aortas (Fig. 4a; Supplementary Fig. 9a), but no change in weight gain, fat mass or blood glucose (Supplementary Fig. 9bd). In vivo delivery of anti-miR-204 prevented HFD-triggered downregulation of aortic Sirt1 (Fig. 4b) and HFD-induced decline of endothelium-dependent vasorelaxation (Fig. 4c) and bioavailable NO (Fig. 4d). In addition, HFD-induced vascular inammation, as assessed by aortic macrophage content, was reduced in mice receiving anti-miR-204 (Fig. 4e,f). Thus, miR-204 mediates impaired endothelial function and vascular inammation secondary to a HFD.
Systemic delivery of miR-204 inhibitor may affect expression of non-vascular miR-204 targets. We therefore examined the effect of miR-204 inhibition on isolated aortas. Ex vivo inhibition of miR-204 with anti-miR-204 in aortas of mice subjected to HFD feeding improved endothelium-dependent vasorelaxation (Fig. 4j), indicating that the extravascular effects of miR-204
NATURE COMMUNICATIONS | 7:12565 | DOI: 10.1038/ncomms12565 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications 3
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12565
a
b
c f
200
#
*
NPD
NPD-AB
NPD-AB-R
400
#
vWF
Sirt1
End. Sirt1
150
miR-204
NFR
Merge
DAPI
350
300
NPD NPD-AB NPD-AB-R
miR-204
100
Trpm3
100
*
*
50
50
0
0
NPD
NPD-AB
NPD-AB-R
NPD
NPD-AB
NPD-AB-R
d
e
g
p-eNOS
110 kDa
38 kDa
250
250
110 kDa
*
eNOS
200
200
*
Normalized to
Gapdh
Sirt1
(IF quantification)
110 kDa
*
Sirt1
Gapdh
150
#
150
#
* #
100
100
#
50
50
NPD
NPD-AB
NPD-AB-R
0
0
Sirt1 eNOS
Aortic Endoth.
NPD NPD-AB NPD-AB-R
i j k
h l
% Contraction (PE 106 M)
100
100
100
100
100
80
80
80
80
80
60
60
60
60
60
40
40
#
40
40
40
20
20
20
20
20
*
* *
0
NPD NPD-AB
NPD-AB-R
0
Sirt1
Sirt1 -AB 0
* *
VE-Cad-CreVE-Cad-Cre-AB 0
eSirt1 eSirt1 -AB
0
#
SC+AdLacZ miR-204+AdLacZ miR-204+AdSirt1
PE 5
6
7
8
9
log (M)A
PE 5
6
7
8
9
log (M)A
PE 5
6
7
8
9
log (M)A
9
8
7
6
5
PE 5
6
PE
9
log (M)A
8
7
log (M)A
Figure 2 | Antibiotics improve vasorelaxation via endothelial Sirt1. (ac) Gut microbiota suppression in mice with broad-spectrum antibiotics downregulates aortic miR-204 (a: qPCR; b: in situ hybridization at 100 magnication with miR-204 shown in purple with red nuclear counterstain, Scale bar;
20 mm) and Trpm3 (c). All data are shown as % of NPD. n 6 for NPD and NPD-AB, n 4 for NPD-AB-R. *Po0.05 versus NPD, #Po0.05 versus NPD-AB.
(d,e) Gut microbiome suppression in mice with broad-spectrum antibiotics upregulates aortic Sirt1 and eNOS expression. All data are shown as % of NPD. n 3 for NPD and NPD-AB, n 4 for NPD-AB-R. *Po0.05 versus NPD, #Po0.05 versus NPD-AB. (f) Immunouorescence of mouse aortic sections showing
upregulation of total aortic and endothelial Sirt1 expression with antibiotics (magnication 63, Scale bar; 20 mm). (g) Quantication of total aortic (n 4
sections) and endothelial (n 15 endothelial cells) Sirt1 immunostaining in f. (h,i) Gut microbiota suppression in mice with antibiotics improves endothelium-
dependent vasorelaxation in wild-type C57Bl/6 (h), VE-Cad-Cre (i) and Sirt1/ (j) mice. NPD: n 10, NPD-AB: n 10, NPD-AB-R: n 8. *Po0.05 versus
NPD, #Po0.05 versus NPD-AB. Sirt1/: n 10, Sirt1/-AB: n 20; *Po0.05 versus Sirt1/. VE-Cad-Cre: n 5, VE-Cad-Cre-AB: n 9, *Po0.05 versus VE
Cad-Cre. (k) Gut microbiome suppression with antibiotics does not improve aortic endothelium-dependent vasorelaxation in eSirt / mice. eSirt1 / :n 14, eSirt1 / AB: n 15. #Po0.05 versus eSirt1 / . (l) Adenovirus-mediated overexpression of Sirt1 lacking 30-UTR rescues miR-204 mimic-induced
impairment of endothelium-dependent vasorelaxation ex vivo. SC-AdLacZ: n 5; miR-204-AdLacZ: n 6; miR-204-AdSirt1: n 6. AdLacZ, control adenovirus-
expressing E. coli LacZ; AdSirt1, adenovirus-expressing Sirt1; SC, scrambled control microRNA. *Po0.05 versus SC-AdLacZ, #Po0.05 versus miR-204-AdLacZ. A, acetylcholine; AB, antibiotics; eSirt1 / , mice conditionally lacking endothelial Sirt1; IF, immunouorescence; NFR, nuclear fast red; NPD, normal pellet diet;
PE, phenylephrine; R, stoppage of antibiotics and recolonization; Sirt1/, control Sirt1 oxed mice. n represents the number of aortic rings in hl. Independent sample t-test was used. Data shown as mean and error bar represents s.e.m.
inhibition do not play a major role in improving vascular endothelial function.
MiR-204 acts primarily via endothelial Sirt1. Because micro-biome-dependent changes in vascular miR-204 and Sirt1 expression were not limited to the endothelium (Figs 2ag and 3af), we more carefully gauged the role of endothelial Sirt1 in improving endothelial function rendered by miR-204 inhibition. Lack of endothelial Sirt1 (eSirt1 / mice) did not completely negate the effect of miR-204 inhibition on HFD-induced endothelial dysfunction, despite a marked decrease in aortic miR-204 (Fig. 4g,h). However, comparison of overall impairment of endothelium-dependent vasorelaxation in wild-type and eSirt1 /
mice indicated that the absence of endothelial Sirt1 signicantly diminishes the benecial impact of miR-204 inhibition on vascular endothelial function (Fig. 4i). Thus, endothelial Sirt1 is a key but not the only target of miR-204 in HFD-induced endothelial dysfunction. Given the nding that neither antibiotics nor miR-204 inhibition impacted endothelium-independent vasorelaxation
(Supplementary Fig. 12), it is likely that targets of miR-204, other than Sirt1, that are partly responsible for miR-204-mediated endothelial dysfunction with HFD feeding are also expressed in the endothelium.
Microbiome-dependent serum factor(s) regulate miR-204. Remote regulation of endothelium-dependent vasorelaxation by the microbiome suggests the presence of second messenger(s) in the systemic circulation that govern expression of vascular miR-204 and Sirt1. Supporting this line of thought, endothelial cells incubated with serum from mice in which the gut micro-biome was suppressed with antibiotics showed decreased miR-204 and Trpm3, and increased Sirt1, expression (Supplementary Fig. 10ac). In contrast, serum from mice in which the micro-biome was repopulated by discontinuation of antibiotics did not downregulate miR-204 in endothelial cells (Supplementary Fig. 10d). In addition, serum from antibiotic-treated mice led to the increase in endothelial Stat3 signalling (Supplementary Fig. 10a).
4 NATURE COMMUNICATIONS | 7:12565 | DOI: 10.1038/ncomms12565 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12565 ARTICLE
a
b
c
Mature miR-204
5,000
Pri-and Pre-
miR-204
*
HFD
HFD-AB
HFD-AB-R
500
4,000
*
400
miR-204
3,000
SC
*
300
2,000
*
miR-204 ISH
quantification
200
1,000
*
#
#
#
100
0
miR-204
NPD
0
NPD
HFD
HFD-AB
HFD-AB-R
NPD
HFD
HFD-AB
HFD-AB-R
NPD
HFD
HFD-AB
HFD-AB-R
d vWF Sirt1
End. Sirt1
Merge
e
f
g
DAPI
HFD-AB HFD-AB-R
NPD HFD HFD-AB HFD-AB-R
150
Sirt1 (IF quantification)
p-Stat3
Sirt1
Gapdh
Gapdh
110 kDa
38 kDa
82 kDa
38 kDa
Total aortic NO x (nitrate +
nitrite) % of NPD
150
NPD HFD
#
#
100
*
100 * # *
#
*
50
50
*
0
NPD
HFD
0
HFD-AB
HFD-AB-R
HFD
HFD-AB
Aortic Endoth.
h
i j
% Nitric oxide bioavailability
(%PE 105 M)
100
250
NPD-SC HFD
HFD-AB HFD-AB-R
# #
WT
% Contraction (PE 106 M)
% Contraction (PE 106 M)
100
80
200
80
60
150
60
40
100
40
20
50
20
eSirt1
NPD
0
NPDHFD HFD-AB HFD-AB-R
0
0
HFD HFD-AB
PE
9
8
7
6
5
9
8
7
6
5
PE
9
8
7
6
5
log (M)A
log (M)PE
log (M)A
Figure 3 | Antibiotics rescue endothelial Stat3 and Sirt1 in high-fat diet-induced vascular dysfunction. (ac) Suppression of gut microbiota with broad-spectrum antibiotics inhibits HFD-induced aortic miR-204. (a) qPCR for mature and precursor miR-204 in mouse aortas. n 4 for NPD and HFD-AB-R, n 6
for HFD and HFD-AB. (b) In situ hybridization for miR-204 in mouse aortas (blue, magnication 100, Scale bar; 20 mm). (c) Quantication of miR-204 in b.
n 5 images from different regions of aortic sections. (df) Suppression of gut microbiome with antibiotics prevents HFD-induced downregulation of aortic
Sirt1 and Stat3 signalling. (d) Immunouorescence for Sirt1 in mouse aortic sections (magnication 63, Scale bar; 20 mm). (e) Quantication of total aortic
(n 4 sections) and endothelial (n 15 endothelial cells) Sirt1 immunostaining in d. (f) Western blot for Sirt1 and phospho-Stat3 in mouse aortas.
Immunoblots were done with pooled aortic samples; n 4 for NPD and HFD-AB-R and n 6 for HFD and HFD-AB. (g) Suppression of gut microbiome
rescues HFD-induced decrease in total aortic NOx [nitrate nitrite]. NPD: n 9, HFD: n 6, HFD-AB: n 6. (h,i) Suppression of gut microbiota rescues HFD-
induced reduction of endothelium-dependent vasorelaxation and nitric oxide bioavailability. NPD: n 10, HFD: n 12, HFD-AB: n 10, HFD-AB-R: n 10. (j)
Suppression of gut microbiota does not rescue HFD-induced impairment of endothelium-dependent vasorelaxation in eSirt1 / mice. eSirt1 / HFD: n 6,
eSirt1 / HFD-AB: n 12. *Po0.05 versus NPD. #Po0.05 versus HFD. A, acetylcholine; AB, antibiotics; eSirt1 / , mice conditionally lacking endothelial
Sirt1; HFD, high-fat diet; NPD, normal pellet diet; PE, phenylephrine; R, stoppage of antibiotics and recolonization. n represents the number of aortic rings in hj. IF, immunouorescence. Independent sample t-test was used. Data shown as mean and error bar represents s.e.m.
The effect of the microbiome on atherosclerosis, glucose homeostasis and adiposity has been attributed, in part, to circulating metabolites such as trimethylamine-oxide and short-chain fatty acids (SCFAs) produced by gut bacteria25,7,20. The receptors for SCFAs, including Gpr41 and 43, and Olfr78, are expressed in the vasculature such as the aorta21, renal arteries21 and myometrial arteries22. We tested whether SCFAs serve as second messengers responsible for the effect of the microbiome on aortic miR-204 and Sirt1 expression, and endothelial function. Supplementation of drinking water with acetate and butyrate, two SCFAs produced by gut bacteria, did not negate the effect of antibiotics on aortic miR-204 expression and endothelium-dependent vasorelaxation (Supplementary Fig. 11a,b). In addition, a mixture of SCFAs did not decrease Sirt1 in endothelial or smooth muscle cells in vitro (Supplementary Fig. 11c,d). The lack of effect of SCFAs suggests that they are not responsible for the communication between the gut microbiome and vascular miR-204.
In summary, these ndings identify a gutvascular axis in which microbes remotely upregulate miR-204 expression in the
vessel wall. Among the genes targeted by miR-204 in the vascular wall is Sirt1, downregulation of which promotes endothelial dysfunction (Fig. 5). In addition, this work demonstrates that nutritional stress in the form of a western diet negatively impacts the endothelium via this same gutvascular axis. Although the data do not identify the mediator(s) responsible for this remote communication between the gut and the vasculature, they do suggest that these messengers are in the systemic circulation, and regulate endothelial miR-204 expression through the Stat3 transcriptional program. It is important to acknowledge that while orally administered antibiotics led to a striking diminution of the gut ora, because a fraction of these antibiotics may be systemically absorbed, their effect on vascular miR-204/Sirt1 expression and endothelial function could also be due to their impact on the composition of the microbiome in niches other than the gut. Finally, given that microbiome-dependent expression of Sirt1 was not limited to the vasculature, and miR-204 was not the only microRNA differentially regulated in the vasculature in germ-free conditions, these ndings may provide a
NATURE COMMUNICATIONS | 7:12565 | DOI: 10.1038/ncomms12565 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications 5
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12565
a
1,000
WT
b
c d
100
250
WT
% Nitric oxide bioavailability
(%PE 105 M)
#
800
*
Sirt1
Gapdh
80
NPD-SC HFD-SC HFD-204-I
200
60
150
miR-204
% Contraction (PE 106 M)
600
150
40
100
400
100
Sirt1
#
20
50
200
NPD-SC HFD-SC HFD-204-I
50
*
#
0
0
0
0
NPD-SC
PE
9
8
7
6
5
9
8
7
6
5
HFD-SC
HFD-204-I
NPD-SC
HFD-SC
HFD-204-I
log (M)A
log (M)PE
f
H & E
4
100
100
NPD-SC HFD-SC HFD-204-I
e
g
300
eSirt 1
*
F4/80
Gapdh
200
600
*
miR-204
Aorta macrophage
(F4/80/Gapdh)
400
L
#
100
200
L
L
#
0
0
F4/80
NPD-SC
HFD-SC
HFD-204-I
NPD-SC
HFD-SC
HFD-204-I
L
L
h
i
*
j
100
*
% Contraction (PE 106 M)
% Impairment in vascular relaxation
(c & h) (% of NPD-SC (WT))
*
50
100
% Contraction (PE 106 M)
80
40
*
80
60
30
60
40
#
#
20
40
eSirt1
20
10
0
20
0
NPD-SC HFD-SC HFD-204-I
NPD-SC
HFD-SC
HFD-204-I
NPD-SC
HFD-SC
HFD-204-I
0
WT
NPD-SC HFD-SC
HFD-204-I
PE
9
8
7
6
5
log (M)A
PE
9
8
7
6
5
WT eSirt1/
log (M)A
Figure 4 | MiR-204 mediates high-fat diet-induced vascular dysfunction and inammation. (a) Systemic delivery of miR-204 inhibitor in HFD-fed mice (a) prevents upregulation of aortic miR-204, (b) downregulation of aortic Sirt1 and (c,d) rescues endothelium-dependent vasorelaxation and bioavailable vascular NO. n 4 for NPD-SC, n 6 for HFD-SC and HFD-204-I in a; n 4 for NPD-SC, n 6 for HFD-SC and HFD-204-I in b; NPD-SC: n 15, HFD-SC: n 16, HFD-
204-I: n 18 in c,d. n represents number of aortic rings. (e,f) Systemic in vivo delivery of miR-204 inhibitor suppresses HFD-induced vascular inammation.
Expression of macrophage marker f4/80 was assessed by immunoblot in aorta (e) and by immunohistochemistry (f) in aortic roots of mice (magnication
4 or 100; scale bars, 500 mm ( 4), 20 mm ( 100)). Adjacent sections of aortic root sections were stained with haematoxylin and eosin (magnication 4 and 100). n 4 for NPD-SC, n 6 for HFD-SC and HFD-204-I, *Po0.05 versus NPD-SC. #Po0.05 versus HFD-SC. HFD, high-fat diet; NPD, normal
pellet diet; SC, scrambled control microRNA; 204-I, miR-204 inhibitor. (gi) Inhibition of miR-204 with HFD feeding improves endothelium-dependent vasorelaxation to a lesser degree in eSirt1 / mice compared with wild-type mice. (g) Aortic expression of miR-204 in eSirt1 / mice with HFD and with miR-204 inhibitor infusion. RTPCR was done with pooled aortic samples. (h) Systemic delivery of miR-204 inhibitor partially improves endothelium-dependent vasorelaxation induced in eSirt1 / mice on HFD. n represents the number of aortic rings. NPD-SC: n 13, HFD-SC: n 12, HFD-204-I: n 9.
(i) Per cent impairment in endothelium-dependent vascular relaxation in c,h. (j) Ex vivo inhibition of miR-204 rescues HFD-induced impairment of endothelium-dependent vasorelaxation. n represents the number of aortic rings. NPD-SC: n 5, HFD-SC: n 10, HFD-204-I: n 10, *Po0.05 versus NPD-SC.
#Po0.05 versus HFD-SC. A, acetylcholine; eSirt1 / , mice conditionally lacking endothelial Sirt1; HFD, high-fat diet; L, lumen; NPD, normal pellet diet; PE, phenylephrine. Independent sample t-test was used. Data shown as mean and error bar represents s.e.m.
6 NATURE COMMUNICATIONS | 7:12565 | DOI: 10.1038/ncomms12565 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12565 ARTICLE
Diet
Gut microbiota
Systemic serum factor
miR-204
Sirt1
Sirt1
Endothelial Dysfunction
Figure 5 | Remote regulation of vascular endothelial function by the microbiome. Gut microbiota-regulated second messengers in the systemic circulation induce vascular miR-204 expression which, in turn, promotes endothelial dysfunction by targeting Sirt1.
broader paradigm for microRNA-mediated regulation of organ physiology and pathophysiology by the microbiome.
Methods
Animals. All animal experiments were approved by Institutional Animal Care and Use Committee and were carried out according to National Institutes of Health (NIH) guidelines. Male GFM and PFM (Swiss-Webster, age 10 weeks) were procured from Taconic, New York, USA. All other studies were performed in C57BL/6, Sirt1/, VE-Cadherin-Cre and endothelium-specic Sirt1 knockout (eSirt1 / )
mice (age 1012 weeks). eSirt1 / mice were generated by crossing Sirt1/ mice with VE-Cadherin-Cre mice. Mice were given either normal pellet diet (NPD) or
HFD. HFD is an adjusted calorie diet that provides 42% calories from fat (TD.88137, Harlan). To suppress the gut ora, mice were removed from the colony, housed separately and were given water supplemented with a cocktail of broad-spectrum antibiotics (metronidazole1 g l 1, ampicillin1 g l 1, neomycin1 g l 1 and vancomycin0.5 g l 1) for a period of 6 weeks. To repopulate antibiotic-treated mice with microbiota, antibiotic water was replaced with normal water and mice were co-housed with their former littermates for a period of 23 weeks. Mice were maintained in specic pathogen-free conditions at the central animal facility of the University of Iowa. The mice receiving cocktail of broad-spectrum antibiotics were monitored for weight loss, diarrhoea and signs of discomfort. Suppression of gut microbiota was conrmed by 16S RNA gene analysis. SCFAs (acetate 90 mM; butyrate 60 mM) were added to drinking water. These concentrations were based on the published work23. Control mice received sodium-matched water. For in vivo infusion of locked nucleic acid microRNA inhibitor, ALZET 2006 osmotic pups containing oligonucleotides (scrambled control or miR-204 inhibitor (miR-204-I)) were aseptically implanted in C57BL/6 or eSirt1 / mice kept on either NPD or
HFD. Mini-osmotic pumps were designed to deliver oligonucleotides at the rate of0.15 ml h 1, and each mouse received oligonucleotides at a dose of B0.7 mg kg 1 per day for a period of 6 weeks.
Reagents. MicroRNA cDNA preparation kit and primers were purchased from Quanta Biosciences Inc., Gaithersburg, MD, USA. Sirt1 (Thermo Scientic-PA5-23-063, Santa Cruz Biotechnology-sc-15404), Gapdh (Trevigen-2275-PC), eNOS(BD Transduction Laboratories-610297), Stat3 (Cell Signalling Technology-4904), phospho- (Tyr-705)-Stat3 (Cell Signalling Technology-9145), F4/80 (abcam-ab6640) antibodies were used.
RNA isolation and RTqPCR. RNA was isolated using Qiazole/Trizole as per the manufacturers instructions. MicroRNAs were converted to cDNA using the qScriptTM microRNA cDNA synthesis kit (Quanta Biosciences). Real-time qPCR was performed using Brilliant II SYBR Green RTqPCR kit. MiR-29b, actin (beta), Rnu6 or Snord47 was used as internal controls. qPCR with reverse transcription (RTqPCR) of Sirt1, Trpm3 and Gapdh were performed using the Brilliant II SYBR Green RTqPCR kit. Gapdh was used as an internal control. Primer sequences are provided in Supplementary Table 1.
NanoString nCounter assay and selection of internal control for qPCR. Total aortic RNA was used as input for the nCounter reactions and was performed
according to the manufacturers instructions. In brief, a unique oligonucleotide was tagged to the microRNAs for the specic and sensitive detection by nCounter analysis system. The system is based on target-specic probe pairs that are hybridized to sample in solution. A reporter probe carries the uorescent signal, while capture probe allows the complex to be immobilized for data collection. The protocol does not involve any amplication steps. The panel was designed to screen 578 mouse microRNAs, 33 viral microRNAs, 3 non-mammalian miRNAs, 8 negative controls and a dilution series of 6 positive controls. nCounter data were normalized for lane-to-lane variation by six dilution series spike positive controls. Global microRNA expression was used to normalize individual microRNA expression. Heat map for expression of microRNAs was prepared using Gene-Pattern (http://genepattern.broadinstitute.org/gp/pages/index.jsf
Web End =http://genepattern.broadinstitute.org/gp/pages/index.jsf). In the absence of universally accepted internal control for normalization of aortic microRNA expression analysis by qPCR, we identied a microRNA that best resembles global microRNA expression as previously described24 with some modications. In brief, to select the microRNA most suitable as a normalization control, the pattern of microRNA expression in PFM and GFM mice normalized with (i) candidate microRNAs and (ii) commonly used normalization mRNAs (Actb (b-actin), B2m (b-2-microglobulin) and Gapdh) was determined and compared with expression pattern as achieved with normalization with global microRNA expression. The data were clustered by pairwise complete linkage based on Pearson correlation using GenePattern (http://genepattern.broadinstitute.org/gp/pages/index.jsf
Web End =http://genepattern.broadinstitute.org/gp/pages/index.jsf). The normalization of microRNAs by miR-29b was found to best resemble the expression prole as achieved with normalization using global microRNA expression, followed by miR-29c, b-actin and Gapdh (Supplementary Fig. 2a), and thus miR-29b was used as an internal control for qPCR experiments in aortic samples. To ascertain change in the expression and to validate selection of internal control for qPCR, a single microRNA (miR-204) was normalized by another common housekeeping gene beta-actin, and the expression of multiple microRNAs was normalized with miR-29b, to see if the pattern observed with nCounter assay remains the same (Supplementary Fig. 2be).
Target gene prediction. MicroRNA-SVR is an algorithm that ranks microRNA mRNA target sites interaction (SVR score). Lower SVR score indicates stronger interaction between a microRNA and mRNA target site25. Differentially regulated microRNAs in GFM were examined for their target genes based on the SVR score (http://www.microRNA.org
Web End =www.microRNA.org, the data are available as tab delimited les). SVR scores of each microRNA for every targeted gene were tabulated at different binding strengths (threshold SVR score o 0.5, o 0.7 and o 0.9), and number of
microRNAs targeting a specic gene was determined. Several microRNAs targeting a specic gene were predicted to be differentially regulated.
In vitro and ex vivo transfection of oligonucleotides. Oligonucleotides were incubated with Lipofectamine2000 (Invitrogen) at room temperature for 20 min before adding to cells in OPTI-MEM (Invitrogen). Four hours later, the medium was replaced by fresh medium and cells were cultured for an additional specied period of time (2472 h). Ex vivo transfection of oligonucleotides into mouse thoracic aorta was performed using Oligofectamine (InVitrogen). Oligos were incubated with Oligofectamine at room temperature for 20 min before adding to aortas in OPTI-MEM (InVitrogen). The aorta was kept in transfection mixture overnight. The sequence of oligonucleotides used in study is provided in Supplementary Table 1.
Luciferase reporter assay. The pRL-CMV and Luciferase-Sirt1-30-UTR plasmids were transfected in the human umbilical vein endothelial cells (HUVECs) with scrambled microRNA or miR-204 mimic (20 pM). Lipofectamine 2000 was used as a transfection agent. The luciferase-to-renilla ratio was calculated to normalize for variations in transfection efciencies.
Ex vivo adenoviral infections. Gene transfer was achieved ex vivo by incubating aortas from mice with 5 108 p.f.u. of the appropriate adenoviral stock at 37 C for
24 h before recording vascular reactivity. For in vitro infections of HUVECs, 100 multiplicity of infection was used. The viruses used were (i) AdLacZ coding for Escherichia coli LacZ, (ii) AdSirt1 coding for Sirt1 and (iii) Ad-DNStat3 coding for non-phosphorylatable dominant-negative Stat3.
Vascular reactivity. Thoracic aortas of mice were used. The animals were rapidly killed by CO2 inhalation. The aorta was carefully dissected, rapidly removed and placed in ice-cold oxygenated Krebs-Ringer bicarbonate solution. The vessel was carefully cleared of loose connective tissue and cut into 510 1.5 mm rings. A single ring from each mouse was suspended between two wire stirrups (150 mm) in a12.5- ml organ chambers of myograph system (DMT Instruments, FL, USA) in 6 ml Krebs-Ringer (95% O25% CO2, pH 7.4, 37 C). One stirrup was connected to a three-dimensional micromanipulator, and the other to a force transducer. The mechanical force signal was amplied, digitalized and recorded (PowerLab 8/30). All concentrationeffect curves were performed on arterial rings beginning at their optimum resting tone. This was determined by stretching arterial rings at 10 min intervals in increments of 100 mg to reach optimal tone (B500 mg). One dose of
NATURE COMMUNICATIONS | 7:12565 | DOI: 10.1038/ncomms12565 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications 7
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12565
KCl (60 mM) was administered to verify vascular smooth muscle viability. Vascular NO bioavailability was determined as the difference in cumulative doseresponse curve for phenylephrine (PE; 10 910 5 M) in the absence or presence of the NO synthase inhibitor L-NAME. Data are presented as per cent change to the maximum contraction induced by PE (10 5 M). Endothelium-dependent and -independent vasorelaxation was determined by generating doseresponse curves to acetylcholine (10 910 5 M) and sodium nitroprusside (10 910 5 M), respectively, on PE (10 6 M)-induced pre-contracted vessels. Vasorelaxation evoked by acetylcholine and sodium nitroprusside was expressed as per cent relaxation, determined by calculating the percentage of inhibition to the pre-constricted tension. Aortic rings not responding to initial dose of KCL or showing auto-relaxation were excluded. Per cent impairment in acetylcholine-induced vascular relaxation was determined by measuring the difference between the mean area under the curve for the NPD-scrambled control group and that of the test groups.
NOx (nitrate/nitrite) measurement. Aortic rings were carefully isolated and immediately frozen. Aorta (57 mm) was used for the nitrate/nitrite assay and was performed as per the manufacturers instructions (Cayman-780001, Ann Arbor, MI, USA).
Immunoblotting. Protein samples were resolved on 1012% SDSpolyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Antigenprimary antibody complexes were incubated with horseradish peroxidase-conjugated secondary antibodies and visualized by western blotting luminol reagent (Thermo, USA). Images were captured and quantied using the Image Lab (BioRad, USA) software, and intensity values were normalized to Gapdh. Uncropped blots are shown in Supplementary Fig. 13.
Histological processing and immunostaining. Sections (5 mm) of formalin-xed parafn-embedded tissues were heated (95 C, 20 min) in citrate buffer (10 mM) for antigen retrieval, followed by incubation with primary antibodies. The working concentration for the primary antibodies were as follows; Sirt1; 1: 200, p-Stat3; 1: 100, vWF; 1:200, anti-f4/80; 1:200. Antigenprimary antibody complexes were probed with polyvalent biotinylated goat anti-rabbit secondary antibody and streptavidinhorseradish peroxidase system to amplify the signals, followed by detection with diaminobenzidine as a chromogen. Slides were counterstained with haematoxylin, dehydrated with alcohols and xylene, and mounted in DPX. Alternatively, antigenprimary antibody complexes were probed with uorescence-tagged secondary antibodies. Aortic root cryosections (20 mM) were examined for anti-F4/80 staining using goat anti-rat secondary antibody and detection with diaminobenzidine. Images were captured using Zeiss confocal microscope (Model 710). Total aortic Sirt1 expression was determined by measuring uorescence intensity using the ImageJ software. To examine the change in endothelial expression of Sirt1, endothelial cells were cropped from the images based on the vWF staining using the Photoshop CC software, and the intensity was determined using the ImageJ software.
In situ hybridization. Deparafnized and rehydrated aortic sections (6 mm) were treated with proteinase K (10 mg ml 1, 5 min, 37 C). Sections were dehydrated using ethyl alcohol and incubated with pre-hybridization buffer, followed by hybridization mixture containing either double digoxigenin(DIG)-tagged miR-204-5p probe or DIG-tagged scrambled probe (36 h, 57 C). Sections were washed with sodium saline citrate and probed with either Dylight 594-labelled anti-DIG goat antibody (working concentration; 1:500, Vector Laboratories, Burlingame, CA, USA) or alkaline phosphatase-conjugated anti-DIG FAB (fragment, antigen-binding) (18 h, 4 C). Following alkaline phosphatase-conjugated FAB binding, BCIP/NBT (5-bromo-4-chloro-30-indolylphosphate/nitro-blue tetrazolium) was used as a chromogen. Slides were counterstained with either Nuclear Fast Red or 4,6-diamidino-2-phenylindole and dehydrated with alcohol and mounted in DPX. Images were captured by charge-coupled device camera attached to the Zeiss microscope (Model BX61). For quantication, slides were counterstained with 4,6-diamidino-2-phenylindole. Six images originating from at least two aortic rings were used for the quantication. The bluepurple colour range was selected using the Photoshop CS6, converted to grey and quantied using the Image J software. Sequence of miR-204-5p double DIG-tagged probe is provided in Supplementary Table 1.
Cell culture. HUVECs (Cat# CC-2519) were purchased from Clonetics(San Diego, CA USA) and cultured in growth factor- and fetal bovine serum-supplemented EGM-2 (Lonza, Walkersville, MD, USA). To examine the effects of mouse serum, 2% fetal bovine serum in EGM-2 was replaced with pooled serum from the specic group of mice.
Statistical analysis. To determine sample sizes, power analysis was carried out using preliminary data sets. Considering the independent sample t-test, a sample size of 5 achieved 490% power for studies involving gene and protein expression.
However, for vascular reactivity experiments, a sample size of 10 achieved 495%
power to reject null hypothesis at the signicance level of 0.05. All the data meet assumptions of the statistical test and different groups had similar variance.
We performed all statistical analysis with SPSS (Version 17.0) unless specied. Signicance of difference between two groups was evaluated using independent sample t-test. Results were considered signicant if P values were r0.05.
Data availability. Microarray data were deposited in the Gene Expression Omnibus database (Accession code GSE64630). The authors declare that the data supporting the ndings of this study are available within the article and from the authors on request.
References
1. Qin, J. et al. A metagenome-wide association study of gut microbiota in type 2 diabetes. Nature 490, 5560 (2012).
2. Backhed, F. et al. The gut microbiota as an environmental factor that regulates fat storage. Proc. Natl Acad. Sci. USA 101, 1571815723 (2004).
3. Wang, Z. et al. Gut ora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature 472, 5763 (2011).
4. Bennett, B. J. et al. Trimethylamine-N-oxide, a metabolite associated with atherosclerosis, exhibits complex genetic and dietary regulation. Cell Metab. 17, 4960 (2013).
5. Koeth, R. A. et al. Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis. Nat. Med. 19, 576585 (2013).
6. Tang, W. H. et al. Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. N. Engl. J. Med. 368, 15751584 (2013).
7. Wang, D. et al. Gut microbiota metabolism of anthocyanin promotesreverse cholesterol transport in mice via repressing miRNA-10b. Circ. Res. 111, 967981 (2012).
8. Davignon, J. & Ganz, P. Role of endothelial dysfunction in atherosclerosis. Circulation 109, III27III32 (2004).
9. Mattagajasingh, I. et al. SIRT1 promotes endothelium-dependent vascular relaxation by activating endothelial nitric oxide synthase. Proc. Natl Acad. Sci. USA 104, 1485514860 (2007).
10. Zhang, Q. J. et al. Endothelium-specic overexpression of class III deacetylase SIRT1 decreases atherosclerosis in apolipoprotein E-decient mice. Cardiovasc. Res. 80, 191199 (2008).
11. Choi, S. E. & Kemper, J. K. Regulation of SIRT1 by microRNAs. Mol. Cells 36, 385392 (2013).
12. Kumarswamy, R. et al. Vascular importance of the miR-212/132 cluster. Eur. Heart J. 35, 32243231 (2014).
13. Menghini, R. et al. MicroRNA 217 modulates endothelial cell senescence via silent information regulator 1. Circulation 120, 15241532 (2009).
14. Saunders, L. R. et al. miRNAs regulate SIRT1 expression during mouse embryonic stem cell differentiation and in adult mouse tissues. Aging (Albany NY) 2, 415431 (2010).
15. Chaudhuri, A. D., Yelamanchili, S. V., Marcondes, M. C. & Fox, H. S. Up-regulation of microRNA-142 in simian immunodeciency virus encephalitis leads to repression of sirtuin1. FASEB J. 27, 37203729 (2013).
16. Zhang, S. et al. MiR-199a-5p promotes migration and tube formation of human cytomegalovirus-infected endothelial cells through downregulation of SIRT1 and eNOS. Arch. Virol. 158, 24432452 (2013).
17. Karali, M., Peluso, I., Marigo, V. & Ban, S. Identication and characterization of microRNAs expressed in the mouse eye. Invest. Ophthalmol. Vis. Sci. 48, 509515 (2007).
18. Courboulin, A. et al. Role for miR-204 in human pulmonary arterial hypertension. J. Exp. Med. 208, 535548 (2011).
19. Xu, G., Chen, J., Jing, G. & Shalev, A. Thioredoxin-interacting protein regulates insulin transcription through microRNA-204. Nat. Med. 19, 11411146 (2013).
20. Backhed, F., Manchester, J. K., Semenkovich, C. F. & Gordon, J. I. Mechanisms underlying the resistance to diet-induced obesity in germ-free mice. Proc. Natl Acad. Sci. USA 104, 979984 (2007).
21. Pluznick, J. L. et al. Olfactory receptor responding to gut microbiota-derived signals plays a role in renin secretion and blood pressure regulation. Proc. Natl Acad. Sci. USA 110, 44104415 (2013).
22. Voltolini, C. et al. A novel antiinammatory role for the short-chain fatty acids in human labor. Endocrinology 153, 395403 (2012).
23. Smith, P. M. et al. The microbial metabolites, short-chain fatty acids, regulate colonic Treg cell homeostasis. Science 341, 569573 (2013).
24. Mestdagh, P. et al. A novel and universal method for microRNA RT-qPCR data normalization. Genome Biol. 10, R64 (2009).
25. Betel, D., Koppal, A., Agius, P., Sander, C. & Leslie, C. Comprehensive modeling of microRNA targets predicts functional non-conserved and non-canonical sites. Genome Biol. 11, R90 (2010).
8 NATURE COMMUNICATIONS | 7:12565 | DOI: 10.1038/ncomms12565 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12565 ARTICLE
Acknowledgements
K.I. was supported by the University of Iowa Endowed Professorship in Cardiovascular Medicine; Q.L. was supported by NIH grant T32 HL007344 and M.K. by NIH grant T32 HL007121. We thank M. Ozaki for the Ad-DNStat3.
Author contributions
A.V. and K.I. conceived the project; A.V., Y.-R.K., S.K., Q.L., M.K. and J.S.J. performed the experiments; K.I. obtained funding; A.V. and K.I. analysed the data and wrote the manuscript.
Additional information
Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications
Web End =http://www.nature.com/ http://www.nature.com/naturecommunications
Web End =naturecommunications
Competing nancial interests: The authors declare no competing nancial interests.
Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/
Web End =http://npg.nature.com/ http://npg.nature.com/reprintsandpermissions/
Web End =reprintsandpermissions/
How to cite this article: Vikram, A. et al. Vascular microRNA-204 is remotely governed by the microbiome and impairs endothelium-dependent vasorelaxation by downregulating Sirtuin1. Nat. Commun. 7:12565 doi: 10.1038/ncomms12565 (2016).
This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the articles Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
Web End =http://creativecommons.org/licenses/by/4.0/
r The Author(s) 2016
NATURE COMMUNICATIONS | 7:12565 | DOI: 10.1038/ncomms12565 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications 9
You have requested "on-the-fly" machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Show full disclaimer
Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer
Copyright Nature Publishing Group Sep 2016
Abstract
Gut microbiota promotes atherosclerosis, and vascular endothelial dysfunction, signalled by impaired endothelium-dependent vasorelaxation, is an early marker of atherosclerosis. Here we show that vascular microRNA-204 (miR-204) expression is remotely regulated by the microbiome, and impairs endothelial function by targeting the Sirtuin1 lysine deacetylase (Sirt1). MiR-204 is downregulated, while Sirt1 is upregulated, in aortas of germ-free mice. Suppression of gut microbiome with broad-spectrum antibiotics decreases miR-204, increases Sirt1 and bioavailable vascular nitric oxide, and improves endothelium-dependent vasorelaxation in mouse aortas. Antibiotics curtail aortic miR-204 upregulation, and rescue decline of aortic Sirt1 and endothelium-dependent vasorelaxation, triggered by high-fat diet feeding. Improvement of endothelium-dependent vasorelaxation by antibiotics is lost in mice lacking endothelial Sirt1. Systemic antagonism of miR-204 rescues impaired endothelium-dependent vasorelaxation and vascular Sirt1, and decreases vascular inflammation induced by high-fat diet. These findings reveal a gut microbe-vascular microRNA-Sirtuin1 nexus that leads to endothelial dysfunction.
You have requested "on-the-fly" machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Show full disclaimer
Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer