Role of NAD(P)H oxidase-derived hydrogen peroxide in regulating inducible nitric oxide synthase expression in interleukin-1beta-stimulated vascular smooth muscle

Mounting evidence indicates that reactive oxygen species (ROS) produced in the vascular wall upon cytokine stimulation play an important regulatory function in cell signaling during vascular injury. This thesis tested the hypothesis that nonphagocytic NADPH oxidase (Nox)-derived H 2O2 regulates the expression of inducible nitric oxide synthase (iNOS) in primary rat vascular smooth muscle cells (VSMC) by modulating the activity of the mitogen activated protein kinases (MAPK). This is based on previous observations that IL-1beta-mediated iNOS expression in VSMC is completely dependent upon extracellular regulated kinases 1 and 2 (ERK1/2) activation. Using confoncal microscopy, we found that IL-1beta rapidly increased intracellular ROS production. Catalase loading inhibited ROS production and potentiated ERK activation and iNOS expression indicating that IL1beta-derived H2O2 negatively regulates iNOS expression through ERK1/2. Catalase loading had no effect on p38MAPK and inhibited activation of one isoform of c-Jun N-terminal kinase (JNK), however the effect of catalase on these MAPKs did not correlate with their role in regulating iNOS expression. Inhibition of p38MAPK potentiated while JNK inhibition partially inhibited iNOS expression. The non-phagocytic homologs of the NAD(P)H oxidase Nox1 and Nox4 were expressed in our VSMC, and Nox4 mRNA was approximately 1300 fold more abundant than Nox1 mRNA in quiescent VSMC. A 24 hour stimulation with IL-1beta caused a 500% increase in Nox1 mRNA and an 82% decrease in Nox4 mRNA levels. Silencing of Nox4 expression with adenoviral expression of siRNA hairpins prior to stimulation reduced IL-1beta induced ROS production and increased iNOS expression which is consistent with the effects of catalase. Nox1 siRNA had no effect on whole cell ROS production which is consistent with its low expression levels. However, silencing Nox1 potentiated iNOS expression indicating Nox1-derived ROS may play a role in cell signaling in spite of being too low to detect. All together, our results suggest that targeting intracellular oxidant producing enzymes in the injured vessel may increase the bioavailability of NO by increasing VSMC iNOS expression.