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. 2011 Mar;31(3):560-6.
doi: 10.1161/ATVBAHA.110.219543. Epub 2011 Jan 4.

α1AMP-activated protein kinase preserves endothelial function during chronic angiotensin II treatment by limiting Nox2 upregulation

Swenja Schuhmacher  1 , Marc Foretz, Maike Knorr, Thomas Jansen, Marcus Hortmann, Philip Wenzel, Matthias Oelze, Andrei L Kleschyov, Andreas Daiber, John F Keaney Jr, Gerhard Wegener, Karl Lackner, Thomas Münzel, Benoit Viollet, Eberhard Schulz
Affiliations

α1AMP-activated protein kinase preserves endothelial function during chronic angiotensin II treatment by limiting Nox2 upregulation

Swenja Schuhmacher et al. Arterioscler Thromb Vasc Biol. 2011 Mar.

Abstract

Objective: Besides its well-described metabolic effects, vascular AMP-activated protein kinase (AMPK) can activate endothelial NO synthase, promotes angiogenesis, and limits endothelial cell apoptosis. The current study was designed to study the effects of α1AMPK deletion during vascular disease in vivo.

Methods and results: Chronic angiotensin II infusion at low subpressor doses caused a mild endothelial dysfunction that was significantly aggravated in α1AMPK-knockout mice. Unexpectedly, this endothelial dysfunction was not associated with decreased NO content, because NO levels measured by serum nitrite or electron paramagnetic resonance were even increased. However, because of parallel superoxide production, NO was consumed under production of peroxynitrite in angiotensin II-treated α1AMPK-knockout mice, associated with NADPH oxidase activation and Nox2 upregulation. As Nox2 is also a component of phagocyte NADPH oxidases, we found a vascular upregulation of several proinflammatory markers, including inducible NO synthase, vascular cell adhesion molecule-1, and cyclooxygenase-2. Cotreatment with the NADPH oxidase inhibitor apocynin was able to prevent vascular inflammation and also partially restored endothelial function in α1AMPK-knockout mice.

Conclusions: Our data indicate that in vivo α1AMPK deletion leads to Nox2 upregulation, resulting in endothelial dysfunction and vascular inflammation. This implicates basal AMPK activity as a protective, redox-regulating element in vascular homeostasis.

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Figures

Figure 1
Figure 1. Effects of ATII on systolic blood pressure and AMPK activity in mouse aorta in vivo
(A) Systolic arterial blood pressure was recorded with the initiation of AT II treatment for up to 7 days via implanted aortic telemetry catheters in α1AMPK−/− and corresponding WT mice. Data are mean + SEM of n=4 animals per group. (B) Aortic homogenates of untreated (Ctl) and ATII treated α1AMPK−/− and corresponding WT mice were homogenized and the lysates corrected for protein content. Immunoblotting was performed using antibodies against total αAMPK, α1AMPK, α2AMPK, threonin172p-AMPK and serine79p-ACC, α-actinin served as a loading control. The shown Immunoblot is representative of 5 independent experiments.
Figure 2
Figure 2. Deletion of α1AMPK enhances endothelial dysfunction but increases vascular NO-production during chronic ATII treatment
(A, B) Endothelial dependent relaxation in response to acetylcholine (ACh) and endothelial independent relaxation in response to nitroglycerin (NTG) were determined by isometric tension studies in intact aortic rings (3mm in length) ex vivo. Data are means ± SEM of n=10 independent experiments. Significance was tested using EC50-values and maximum relaxation; * indicates p<0.05 vs. untreated WT; ‡ indicates p<0.05 vs. ATII-treated WT mice (WT + ATII). Vascular NO production was determined by 2 independent assays. (C) First, serum nitrite levels were determined using an NO analyzer, data are means ± SEM of n=6. (D, E) Aortic NO production was also measured using electron paramagnetic resonance spectroscopy. Intensity (I) of the characteristic Fe(DETC)2 EPR signal reflects amount of NO produced by vascular segment during 1hr. Representative spectra are shown in (D), summarized data as means ± SEM (arbitrary units (AU) per [mg] dry weight) n=3 in (E). * indicates p<0.05 vs. untreated WT, ‡ indicates p<0.05 vs. untreated α1AMPK−/−. (F) Immunoblotting was performed in aortic homogenates using total eNOS and serine1177-phospho-eNOS antibodies. The immunoblot shown is representative of 5 independent experiments, * indicates p<0.05 vs. untreated WT, ‡ indicates p<0.05 vs. untreated α1AMPK−/−.
Figure 3
Figure 3. α1AMPK knockout mice display increased vascular oxidative stress and peroxynitrite formation
(A) Aortic homogenates were transferred onto a nitrocellulose membrane by using dot blot technique and incubated with a nitrotyrosine specific antibody. Dot blot shown is representative of n=10 independent experiments, bar graphs were obtained by densitometric analysis. * indicates p<0.05 vs. untreated WT, ‡ indicates p<0.05 vs. ATII treated WT. (B) Immunostaining of aortic sections by using a mouse monoclonal 3-nitrotyrosine antibody (brown colour, intense staining in the endothelium and adventitia of ATII-treated α1AMPK−/− mice is shown by black arrows; white arrows indicate localization of the external elastic lamina) was performed as a marker of local peroxynitrite formation. Results are representative of n=3 experiments. (C) Vascular peroxynitrite / superoxide formation was assessed in intact aortic rings 3mm in length by L-012-enhanced chemiluminescence. Data are mean ± SEM of n=8, * indicates p<0.05 vs. untreated WT, ‡ indicates p<0.05 vs. ATII-treated WT (WT + ATII). (D) Transverse aortic cryosections were labeled with dihydroethidium (DHE; 1 μmol/L), which produces red fluorescence when oxidized to ethidium by superoxide. “E” indicates the endothelium (points to the right), “A” indicates the adventitia, lamina autofluorescence is green. Data are representative of n=4.
Figure 4
Figure 4. Deletion of α1AMPK increases vascular NADPH oxidase activity and Nox-2 mRNA and protein expression
(A) Vascular NADPH oxidase activity was analyzed using aortic membrane preparations and quantified by cytochrome C reduction. Data are means ± SEM of n=6, * indicates p<0.05 vs. untreated WT, ‡ indicates p<0.05 vs. ATII-treated WT mice (WT + ATII). (B) Aortic mRNA expression of NADPH oxidase subunits p22phox, Nox1 and Nox2 was examined by reverse transcription real-time PCR (qRT-PCR). Data are means ± SEM of n=4, ‡ indicates p<0.05 vs. ATII-treated WT. (C, D) A representative Nox2 Immunoblot with densitometric analysis and Nox2 immunostaining (brown color, intense staining is indicated by black arrows; white arrows indicate localization of the external elastic lamina) from aortic tissue is displayed. The endothelial layer points to the left side. Representative sections of n=3 animals per group are shown. The immunoblot shown is representative of 8 independent experiments, the bar graphs were obtained after densitometric analysis, ‡ indicates p<0.05 vs. untreated α1AMPK−/−.
Figure 5
Figure 5. α1AMPK knockout enhances vascular inflammation caused by treatment with subpressor doses of ATII
(A–C) Aortic mRNA expression of iNOS, COX-2 and VCAM-1 was determined by reverse transcription real-time PCR. (D) For vascular iNOS, COX-2 and VCAM-1 protein expression, a representative immunoblot is shown. PCR data are means ± SEM of n=4, immunoblot data are means ± SEM of n=6, ‡ indicates p<0.05 vs. ATII-treated WT (WT + ATII), * indicates p<0.05 vs. untreated WT.
Figure 6
Figure 6. In vivo NADPH oxidase inhibition prevents vascular inflammation and improves endothelial function in α1AMPK knockout mice
α1AMPK−/− mice were treated with low dose angiotensin II +/− the NADPH oxidase inhibitor apocynin for 7 days. (A) Endothelial function was assessed by isometric tension studies ex vivo using intact aortic rings. (B) Representative DHE staining of aortic cryosections. “E” indicates the endothelium, “A” indicates the adventitia, lamina autofluorescence is green. (CE) As a surrogate of vascular inflammation, the mRNA expression of iNOS (C), COX-2 (D) and VCAM-1 (E) was detected in aortic tissue by reverse transcription real time PCR. Data are mean ± SEM of n=4–8 independent experiments, ** indicates p<0.05 vs. untreated α1AMPK−/−, ‡ indicates p<0.05 vs. AT II-treated WT mice, § indicates p<0.05 vs. AT II-treated α1AMPK−/−.
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