P108	Proteasome inhibition improves TNFα-induced endothelial dysfunction.
N.Wilck, M.Lorenz, A.Ludwig, S.Meiners, G.Baumann, V.Stangl, K.Stangl
Kardiologisches Forschungslabor, CCM, Charite-Universitätsmedizin Berlin, Berlin, DE.

The ubiquitin-proteasome system is the major pathway for protein degradation in eukaryotic cells. Recently we have shown that inhibition of the proteasome with MG132 increased endothelial nitric oxide synthase (eNOS) expression and activity in endothelial cells. The aim of our study was to investigate whether MG132 is capable of improving TNFalpha-induced endothelial dysfunction. Endothelium-dependent (acetylcholine) and endothelium-independent (papaverine) vasorelaxation was performed in phenylephrine-precontracted rat aortic rings. Compared to control, in vitro incubation of the rings with TNFalpha for 48 h resulted in a significant dose-dependent reduction of acetylcholine-induced vasorelaxation by 34±9% (250 pg/ml) and 49±8% (500 pg/ml). Co-incubation with TNFalpha (250 pg/ml) and MG132 (50 and 100 nM) for 48 h resulted in a dose-dependent improvement of endothelium-dependent vasorelaxation (22,9±6% for 50 nM MG132; 35±5,9% for 100 nM MG132) in comparison to TNFalpha-treated rings. Endothelium-independent vasorelaxation remained unaffected in all rings. To explore the underlying mechanisms we analysed mRNA levels of important determinants of vascular function by real-time RT-PCR. MG132 significantly increased SOD1 mRNA expression and reduced mRNA levels of several subunits of the NAD(P)H oxidase complex: gp91phox, Nox4, p47phox, p40phox, p22phox. Consequently, superoxide production was reduced in rings incubated with 100 nM MG132 in comparison to controls as shown by lucigenin chemiluminescence. Interestingly, mRNA expression of the potent vasoconstrictor endothelin-1 was upregulated by TNFalpha and markedly decreased in rings incubated with MG132. eNOS mRNA levels in rat aortic rings did not correlate with their improved vascular function after treatment with MG132. In conclusion, low-dose proteasome inhibition with MG132 improves vascular function in an aortic ring model of TNFalpha-induced endothelial dysfunction. By reducing the expression of the NAD(P)H oxidase complex and by enhancing the expression of the antioxidant SOD, subsequent amelioration of NO bioavailability is presumably one underlying mechanism for MG132-induced improvement in endothelial function. Moreover, by suppression of endothelin-1 mRNA levels in MG132 treated rings the balance between vasoconstriction and vasodilatation is shifted in favour of endothelium-dependent vasodilatation.

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