Mycophenolate Acid Inhibits Endothelial NAD(P)H Oxidase Activity and Superoxide Formation by a Rac1-Dependent Mechanism

Abstract

Abstract-Endothelial dysfunction precedes hypertension and atherosclerosis and predicts cardiac allograft vasculopathy and death in heart transplant recipients. Endothelial overproduction of reactive oxygen species, such as superoxide anions produced by NAD(P)H oxidase, induces endothelial dysfunction. Because immunosuppressive drugs have been associated with increased reactive oxygen species production and endothelial dysfunction, we sought to elucidate the underlying mechanisms. Reactive oxygen species, release of superoxide anions, and NAD(P)H oxidase activity were studied in human umbilical vein endothelial cells and in polymorphonuclear neutrophils. Gp91ds-tat was used to specifically block NAD(P)H oxidase. Transcriptional activation of different subunits of NAD(P)H oxidase was assessed by real-time RT-PCR. Rac1 subunit translocation and activation were studied by membrane fractionation and pull-down assays. Calcineurin inhibitors significantly increased endothelial superoxide anions production because of NAD(P)H oxidase, whereas mycophenolate acid (MPA) blocked it. MPA also attenuated the respiratory burst induced by neutrophil NAD(P)H oxidase. Because transcriptional activation of NAD(P)H oxidase was not affected, but addition of guanosine restored endothelial superoxide anions formation after MPA treatment, we speculate that the inhibitory effect of MPA was mediated by depletion of cellular guanosine triphosphate content. This prevented activation of Rac1 and, thus, of endothelial NAD(P)H oxidase. Because all heart transplant recipients are at risk for cardiac allograft vasculopathy development, these differential effects of immunosuppressants on endothelial oxidative stress should be considered in the choice of immunosuppressive drugs.

Mycophenolic acid in Rheumatology: mechanisms of action and severe adverse events

“…In addition, MPA reduces endothelin-1 (ET-1) expression (22) and increases prostacyclin (PGI 2 ) release (23); it suppresses the cytokine-induced production of nitric oxide (NO) (24), presumably because of lack of GTP-derived tetrahydrobiopterin, essential coenzyme of the inducible NO synthetase (iNOS). Moreover, MPA (1-10 µM) inhibits superoxide anion production by endothelial NADPH-oxidase (Nox), since GTP depletion leads to the inactivation of Rac1, a G-protein involved in Nox activity (25). Instead, the effects on ICAM-1 and IL-6 seem to be IMPDH-independent (21).…”

“…MPA (1-10 µM) also inhibits superoxide anion and hydrogen peroxide production in neutrophils activated in vitro, because of Nox activation impairment by Rac1 or Protein kinase C (PKC) (25,33). Nevertheless, an increase in hydrogen peroxide production was even observed after 30 minutes from neutrophil activation; that might explain the paradoxical acute inflammatory syndrome rarely reported in patients treated with MMF (34-35).…”

Cytotoxicity of paraquat in microglial cells: Involvement of PKCδ- and ERK1/2-dependent NADPH oxidase

“…Due to interference of DPI with MTT, its effect on paraquat-induced cytotoxicity was not determined (O'Donnell et al, 1993). We also tested the effects of gp91ds-tat, a specific peptide inhibitor for NADPH oxidase, (Krotz et al, 2007). However, for unknown reasons, this peptide compound failed to work in microglial cells (data not shown).…”

Mycophenolic Acid Is a New Nox2 Inhibitor

“…In conclusion, Krötz et al 1 showed that the propensity of immunosuppressors to induce allograft vasculopathy seems to be correlated with their induction of Nox-mediated superoxide production in ECs, suggesting that Nox inhibitors could be used to correct this serious adverse effect of calcineurin inhibitors. MPA, in contrast to calcineurin inhibitors, reduced reactive oxygen species generation by inhibition of Rac1 and subsequent Nox2 activation in ECs.…”

“…However, vasculopathy remains a major cause of long-term graft failure; thus, a better understanding of its mechanisms is required to improve tolerance. Because endothelial dysfunction is an early manifestation of vascular disorders, Krötz et al, 1 in this issue of Hypertension, hypothesized that endothelial cells (ECs) may be directly affected by immunosuppressive treatment. Therefore, they tested 3 major classes of these drugs with distinct mechanisms of action and effects on the vasculature.…”