Reversal of Endothelial Nitric Oxide Synthase Uncoupling and Up-Regulation of Endothelial Nitric Oxide Synthase Expression Lowers Blood Pressure in Hypertensive Rats
Huige Li, MD, PhD*,*,
Klaus Witte, MD ,
Michael August, MS ,
Isolde Brausch, BS*,
Ute Gödtel-Armbrust, BS*,
Alice Habermeier, BS*,
Ellen I. Closs, PhD*,
Mathias Oelze, PhD,
Thomas Münzel, MD, PhD and
Ulrich Förstermann, MD, PhD*
* Department of Pharmacology, Johannes Gutenberg University, Mainz, Germany
Department of Internal Medicine II, Johannes Gutenberg University, Mainz, Germany
Institute of Pharmacology and Toxicology, Faculty of Clinical Medicine Mannheim, Ruprecht Karls University Heidelberg, Mannheim, Germany
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Figure 1 Midostaurin reduces aortic production of reactive oxygen species and reverses endothelial-type nitric oxide synthase (eNOS) uncoupling in spontaneously hypertensive rats (SHR). Aortas were isolated from SHR or Wistar-Kyoyo (WKY) rats treated orally with vehicle or midostaurin (+Mido, 75 mg/kg/day for 5 days). Production of reactive oxygen species (ROS) from aortic rings (3 mm) was determined by L-012–derived chemiluminescence in the absence or presence of the NOS inhibitor L-NAME (1 mmol/l) (A). Each column represents ROS determinations (mean ± SEM) obtained with aortas from six different animals (**p < 0.01 compared with any other column, tested by analysis of variance (ANOVA) followed by Fisher protected least-significant-difference test). (B) ROS production in SHR aorta as detected with fluorescent microscopy using dihydroethidium. Aortas were incubated with or without L-NAME (1 mmol/l) and then labeled with dihydroethidium, which produces a red fluorescence when oxidized to ethidium by superoxide. The autofluorescence of the elastic laminae is in green. Arrows indicate the endothelium.
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Figure 2 Midostaurin reduces the messenger ribonucleic acid (mRNA) expression of the NADPH oxidase subunit Nox1 in SHR. Total RNA was isolated from the aortas of 18 SHR and 12 WKY (receiving vehicle or 75 mg/kg/day midostaurin for 5 days, +Mido). Quantitative real-time polymerase chain reaction was performed to analyze the mRNA expression of the NADPH oxidase (Nox) subunits Nox1, Nox2 (gp91phox), Nox4, and p22phox. Each column represents data (mean ± SEM) obtained with aortas from six to nine different animals (**p < 0.01, ***p < 0.001, tested by ANOVA followed by Fisher protected least-significant-difference test). Abbreviations as in Figure 1.
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Figure 3 Midostaurin increases the vascular content of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) in SHR. BH4 levels were measured by high-performance liquid chromatography in aortas from 18 SHR and 12 WKY (receiving vehicle or 75 mg/kg/day midostaurin for 5 days, +Mido). Each column represents data (mean ± SEM) obtained with aortas from six to nine different animals (*p < 0.05, ***p < 0.001, tested by ANOVA followed by Fisher protected least-significant-difference test). Abbreviations as in Figure 1.
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Figure 4 Midostaurin increases eNOS mRNA expression in the aorta of SHR and WKY rats. Rats were treated orally with vehicle or midostaurin (+Mido, 75 mg/kg/day) for 5 days. Endothelial nitric oxide synthase mRNA expression in aortas was analyzed with RNase protection assay. Panels A and C show representative gels of an RNase protection assay. Panels B and D show the results of densitometric analyses. Each column (mean ± SEM) represents data obtained with aortas from nine different rats (***p < 0.001 compared vehicle, tested by ANOVA followed by Fisher protected least-significant-difference test). Abbreviations as in Figures 1 and 2.
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Figure 5 Midostaurin increases eNOS protein expression in the aorta of SHR. Six SHR were treated orally with vehicle (SHR) and six other SHR with midostaurin (SHR+Mido, 75 mg/kg/day) for 5 days. Endothelial nitric oxide synthase was stained immunohistochemically with an anti-eNOS monoclonal antibody. In the negative control, mouse immunoglobulin G was used instead of the anti-eNOS antibody. Micrographs are shown in 400x magnification. Abbreviations as in Figure 1.
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Figure 6 Midostaurin increases NO production in the aorta and serum levels of nitrite/nitrate in SHR and WKY. SHR and WKY were treated orally with vehicle or midostaurin (+Mido, 75 mg/kg/day) for 5 days. Bioactive NO in SHR aorta was assessed by electron paramagnetic resonance spin trapping technique using colloid Fe(DETC)2. Serum nitrite/nitrate was measured with an NO analyzer. (A) Original spectra. Arrows indicate the typical triplet caused by NO-Fe(DETC)2. (B) Quantification of NO-trapping experiments from the aortas of 12 animals normalized to aortic area and incubation time. (C) Serum levels of nitrite/nitrate in 36 SHR and 24 WKY. Columns represent mean ± SEM (***p < 0.001, tested by ANOVA followed by Fisher protected least-significant-difference test). Abbreviations as in Figure 1.
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Figure 7 Midostaurin enhances vasodilator response of SHR aorta. SHR were treated orally with vehicle (Co) or midostaurin (Mido, 75 mg/kg/day) for five days and aortic rings were prepared. Vasodilator response to acetylcholine was assayed in organ chambers after precontraction with 100 nmol/l norepinephrine in the absence or presence of the NOS inhibitor L-NAME (1 mmol/l). Symbols represent mean ± SEM of experiments performed with nine different aortas each (**p < 0.01, compared with Co, tested by ANOVA for repeated measures). Abbreviations as in Figure 1.
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Figure 8 Midostaurin lowers blood pressure in SHR and WKY, but not in L-NAME–treated WKY. Systolic and diastolic blood pressure (SBP, DBP) was measured telemetrically in conscious rats with implanted transmitter-coupled pressure transducers. (A) Spontaneously hypertensive rats were first treated orally with vehicle for 1 week. After recovery for another week, rats were treated orally with midostaurin (Mido, 75 mg/kg/day) for 7 days. Arrows indicate the single doses. Symbols represent mean ± SEM of three experiments of six rats each. (B) Wistar-Kyoto rats were treated orally with midostaurin (Mido, 75 mg/kg/day) for 7 days. Symbols represent mean ± SEM of nine rats. (C) Wistar-Kyoto rats were treated with the NOS inhibitor L-NAME (24 mg/kg/day, in the drinking water) from day 2 through day 7. Midostaurin (Mido, 75mg/kg/day) was administrated orally from day 4 through day 7. Symbols represent mean ± SEM of 9 rats. (*p < 0.05, **p < 0.01 ***p < 0.001, compared with the value before midostaurin treatment, tested by ANOVA followed by Fisher protected least-significant-difference test.) Abbreviations as in Figure 1.
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