Gut Microbiota Promote Angiotensin II-Induced Arterial Hypertension and Vascular Dysfunction - PubMed (original) (raw)
. 2016 Aug 30;5(9):e003698.
doi: 10.1161/JAHA.116.003698.
Tanja Schönfelder 2, Ines Brandão 2, Eivor Wilms 2, Nives Hörmann 2, Sven Jäckel 2, Rebecca Schüler 3, Stefanie Finger 2, Maike Knorr 1, Jeremy Lagrange 2, Moritz Brandt 1, Ari Waisman 4, Sabine Kossmann 1, Katrin Schäfer 1, Thomas Münzel 5, Christoph Reinhardt 6, Philip Wenzel 7
Affiliations
- PMID: 27577581
- PMCID: PMC5079031
- DOI: 10.1161/JAHA.116.003698
Gut Microbiota Promote Angiotensin II-Induced Arterial Hypertension and Vascular Dysfunction
Susanne H Karbach et al. J Am Heart Assoc. 2016.
Abstract
Background: The gut microbiome is essential for physiological host responses and development of immune functions. The impact of gut microbiota on blood pressure and systemic vascular function, processes that are determined by immune cell function, is unknown.
Methods and results: Unchallenged germ-free mice (GF) had a dampened systemic T helper cell type 1 skewing compared to conventionally raised (CONV-R) mice. Colonization of GF mice with regular gut microbiota induced lymphoid mRNA transcription of T-box expression in T cells and resulted in mild endothelial dysfunction. Compared to CONV-R mice, angiotensin II (AngII; 1 mg/kg per day for 7 days) infused GF mice showed reduced reactive oxygen species formation in the vasculature, attenuated vascular mRNA expression of monocyte chemoattractant protein 1 (MCP-1), inducible nitric oxide synthase (iNOS) and NADPH oxidase subunit Nox2, as well as a reduced upregulation of retinoic-acid receptor-related orphan receptor gamma t (Rorγt), the signature transcription factor for interleukin (IL)-17 synthesis. This resulted in an attenuated vascular leukocyte adhesion, less infiltration of Ly6G(+) neutrophils and Ly6C(+) monocytes into the aortic vessel wall, protection from kidney inflammation, as well as endothelial dysfunction and attenuation of blood pressure increase in response to AngII. Importantly, cardiac inflammation, fibrosis and systolic dysfunction were attenuated in GF mice, indicating systemic protection from cardiovascular inflammatory stress induced by AngII.
Conclusion: Gut microbiota facilitate AngII-induced vascular dysfunction and hypertension, at least in part, by supporting an MCP-1/IL-17 driven vascular immune cell infiltration and inflammation.
Keywords: angiotensin II; arterial hypertension; gut microbiota; inflammation; monocytes; vascular dysfunction.
© 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.
Figures
Figure 1
Germ‐free (GF) mice are protected from Ang
II
‐induced vascular oxidative stress and inflammatory gene expression.
CONV
‐R versus
GF
mice on C57
BL
/6 background±Ang
II
(1 mg/kg per day) were studied after 7 days of in vivo treatment. A, Aortic superoxide formation. Left: Representative dihydoethidium photomicrotopographs of aortic cryosections; superoxide formation appears in red. Right: Quantification, n=3 to 6 mice per group, Kruskal–Wallis test with Dunn′s multiple comparison. B, Respiratory burst in whole blood in the presence of the phorbolester,
PDB
u (100 nmol/L), measured by L012 (100 μmol/L)
ECL
. 1‐way
ANOVA
with Bonferroni post‐hoc test, n=5 to 13 mice per group. C, Aortic Ror‐γt and
GATA
‐3
mRNA
expression. Kruskal–Wallis test with Dunn′s multiple comparison of selected columns (Rorγt: n=3 to 10 mice per group,
CONV
‐R vs
CONV
‐R+Ang
II
and
GF
vs
GF
+Ang
II
were compared.
GATA
‐3: n=3 to 8 mice per group,
CONV
‐R+Ang
II
and
GF
+Ang
II
were compared). D, Aortic
iNOS
and Nox2
mRNA
expression. Kruskal–Wallis test with Dunn′s multiple comparison,
iNOS
: n=4 to 12 mice per group, Nox2: n=4 to 12 mice per group. AngII indicates angiotensin II; CONV‐R, conventionally raised; ECL, enhanced chemiluminescence; GATA‐3, GATA binding protein 3; iNOS, inducible nitric oxide synthase; Nox2, NADPH oxidase 2; Rorγt, retinoic acid‐related orphan nuclear receptor gamma t.
Figure 2
Absence of gut microbiota attenuates Ang
II
‐induced vascular infiltration of myelomonocytic cells into the aortic vessel wall.
CONV
‐R versus
GF
mice on C57
BL
/6 background±Ang
II
(1 mg/kg per day) were studied after 7 days of in vivo treatment. A, Aortic Ccl‐2
mRNA
expression. Kruskal–Wallis test with Dunn′s multiple comparison test, n=4 to 12 mice per group. B, Intravital videomicroscopy imaging of rolling leukocyte in carotid arteries of Ang
II
‐infused
GF
and
CONV
‐R mice. Left panel, representative pictures; right panel, quantification. Kruskal–Wallis test with Dunn′s multiple comparison test, n=3 to 5 mice per group. C, Flow cytometric analysis of total
CD
45.2+ cells per aorta. Kruskal–Wallis test with Dunn′s multiple comparison test, n=10 to 11 mice per group. D, Flow cytometric analysis of
CD
11b+ cells as well as
CD
11b+Ly6G+ and
CD
11b+Ly6G−Ly6C+ cells per aorta. Pregating on living
CD
45+ cells. Total cell numbers are shown in bar graphs. Kruskal–Wallis test with Dunn′s multiple comparison test, n=10 to 12 mice per group. Below, representative fluorescence‐activated cell sorting plots are given. AngII indicates angiotensin II; Ccl‐2, chemokine (C‐C motif) ligand 2; CONV‐R, conventionally raised; GF, germ‐free.
Figure 3
Germ‐free (GF) mice are protected from Ang
II
‐induced vascular dysfunction and blood pressure increase.
CONV
‐R versus
GF
mice on C57
BL
/6 background±Ang
II
(1 mg/kg per day) were studied after 7 days of in vivo treatment. A and B, Cumulative concentration relaxation curves of isolated aortic rings in response to endothelium‐dependent (
AC
h) and endothelium‐independent vasodilatators (
GTN
). Two‐way
ANOVA
, both n=6 to 15 mice per group. *P<0.05 versus
CONV
‐R; † P<0.05 versus
GF
; ‡ P<0.05 versus
GF
+Ang
II
. C, Blood pressure of
CONV
‐R and
GF
mice with and without Ang
II
challenge, measured by tail‐cuff method. Kruskal–Wallis test with Dunn′s multiple comparison, n=4 to 5 mice per group. D, Flow cytometric analysis of kidneys of
CONV
‐R control mice and
GF
mice with and without Ang
II
treatment.
CD
45+ as well as
CD
11b+ cells (pregated on living
CD
45+ cells) per kidney in
CONV
‐R control mice and
GF
mice±Ang
II
treatment are shown in bar graphs. Statistical analysis was performed with 1‐way
ANOVA
with Bonferroni post‐hoc test, n=5 to 8 mice per group. Below, representative fluorescence‐activated cell sorting plots are given.
AC
h indicates acetylcholine; AngII, angiotensin II; CONV‐R, conventionally raised; GTN, glyceryl trinitrate.
Figure 4
Ang
II
‐triggered cardiac fibrosis, as well as accumulation of myelomonocytic cells, is attenuated in
GF
mice.
CONV
‐R versus
GF
mice±Ang
II
(1 mg/kg per day) were studied after 7 days of in vivo treatment. A, Cardiac sections were stained with Masson's trichrome (
MTC
) staining to assess fibrosis and analyzed by immunohistochemistry staining for
MAC
‐2+ as well as
ELANE
- cells to assess macrophage as well as neutrophil accumulation, respectively. B, Representative images of cardiomyocyte cross‐sectional areas of cardiac sections.
FITC
‐labeled wheat germ agglutinine probed cardiac membranes appear in green. C, Quantification.
MAC
‐2,
ELANE
, and
MTC
: Kruskal–Wallis test with Dunn′s multiple comparison of selected columns (
CONV
‐R vs
CONV
‐R Ang
II
,
GF
vs
GF
Ang
II
), n=3 to 4 mice per group. Cardiomyocyte (CM) area: Kruskal–Wallis test with Dunn′s multiple comparison of selected columns (
GF
vs
GF
Ang
II
,
CONV
‐R Ang
II
vs
GF
Ang
II
), n=3 to 4 mice per group. AngII indicates angiotensin II; CONV‐R, conventionally raised; GF, germ‐free.
Figure 5
End‐organ damage in Ang
II‐
induced hypertension is attenuated in GF mice.
CONV
‐R versus
GF
mice±Ang
II
(1 mg/kg per day) were studied after 7 days of in vivo treatment. A through C, Left ventricular (LV) ejection fraction (
LVEF
; %),
LV
mass (g), as well as intraventricular septum (IVS) thickness at diastole (mm) were measured and delineated by high‐frequency ultrasound. Kruskal–Wallis test with Dunn′s multiple comparison. n=4 mice per group; *P<0.05. D, Representative ultrasound pictures including
IVS
, left‐ventricular internal diameter (
LVID
), as well as the left‐ventricular posterior wall (
LVPW
) at diastole (over 500 ms). AngII indicates angiotensin II; CONV‐R, conventionally raised; GF, germ‐free.
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