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Background: A functional endothelium protects against atherosclerosis and thrombosis. Cardiovascular diseases are often associated with oxidant stress involving protein kinase C (PKC) and uncoupling of endothelial NO synthase (eNOS). Methods and Results: The expression of NADPH oxidases isoforms (Nox1, Nox1, Nox4 and p22phox) was significantly higher in the aorta of Spontaneously hypertensive rats (SHR) compared with Wistar-Kyoto (WKY) rats (quantitative real-time RT-PCR, qRT-PCR). Aortas of SHR showed an elevated production of reactive oxygen species (ROS) relative to WKY rats (L-012 chemiluminescence and dihydroethidium fluorescence). The aortic production of ROS in SHR was partially reduced by the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME), indicating eNOS uncoupling (i.e. eNOS as a source of ROS) in this vascular pathology model. Oral treatment of SHR with the PKC inhibitor midostaurin (4'-N-benzoyl staurosporine, CGP 41251, PKC-412) led to a marked reduction of aortic Nox1expression and a marked reduction of aortic ROS production and reversed eNOS uncoupling. The aortic levels of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4), an essential cofactor of eNOS, was elevated by Midostaurin. In SHR, bioactive NO in the aorta and nitrite/nitrate in plasma increased in response to midostaurin. Interestingly, midostaurin treatment also increased the vascular expression of eNOS mRNA and protein. Ex vivo, aortas from midostaurin-treated SHR and WKY rats showed an enhanced NO-mediated relaxation in response to acetylcholine. Treatment with midostaurin lowered blood pressure in SHR, but not in WKY rats made equally hypertensive with L-NAME. Conclusions: PKC inhibition in SHR downregulates Nox1, reduces oxidative stress, and increases vascular BH4 levels. This reverses eNOS uncoupling. The parallel upregulation of eNOS gene expression enhances the amount of functional enzyme, produces more bioactive NO, and lowers blood pressure.
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J. Vasc. Biol. 42, Sup:2 (2005) p32