NADPH oxidases are responsible for the failure of nitric oxide to inhibit migration of smooth muscle cells exposed to high glucose

Abstract

Our previous studies showed that nitric oxide (NO) fails to inhibit migration of smooth muscle cells (SMC) exposed to high glucose (HG) because of oxidation of the most reactive cysteine, cysteine-674, on the sarco/endoplasmic reticulum ATPase, preventing its S-glutathiolation, thus blocking NO action. This study further addresses the sources of the oxidants responsible for the failure of NO to inhibit SMC migration in HG. NADPH oxidases are the major source of reactive oxygen species (ROS) in SMC. We used small interfering RNA or dominant-negative adenoviral vectors to target components of NADPH oxidase to study their individual roles by measuring serum-induced migration in the presence or absence of NO. In HG, the mRNA levels of Nox1 and Nox4 and the protein level of Nox4 were increased; knocking down Nox1 or Nox4 attenuated the ROS production and restored the inhibition of SMC migration by NO. Blockade of the activation of Rac1 or p47(phox) inhibited serum-induced migration and restored the inhibition of migration by NO. These data indicate that NADPH oxidases are responsible for the failure of NO to inhibit SMC migration caused by HG.

Figure 1

The effect of high glucose on expression levels of NADPH oxidase in SMC. A. The mRNA levels of NADPH oxidase components by real time quantitative PCR. *P<0.05, paired t-test between cells treated with normal glucose (NG) and high glucose (HG). N=5. B. HG increased Nox4 protein level compared with NG. *P<0.05, N=3.

Figure 2

Osmolarity is not involved in the increased high glucose-induced ROS production. A&B. High concentration of mannose (19.5 mM mannose plus 5.5 mM glucose) didn't increase Nox1 and Nox4 mRNA levels compared with normal glucose. N=5, one-way ANOVA. *P<0.05 compared with normal glucose. C. High glucose increased ROS production compared with normal glucose. N=3, one-way ANOVA. *P<0.05 compared with normal glucose.

Figure 3

Nox1 and Nox4 mediate high glucose induced ROS production and abnormal response to NO in SMC. A. The Nox1 and Nox4 mRNA levels were knocked down individually with their respective siRNA in SMC cultured in HG. *P<0.05 compared with control siRNA, N=3. B. Knocking down Nox1 or Nox4 attenuated the ROS production induced by HG. *P<0.05 compared with the NG group, N=4, one-way ANOVA. C. The application of either Nox1 or Nox4 siRNA restored the IL-1β induced inhibition of cell migration in HG. *P<0.05 compared cells treated with serum alone, N=6, one-way ANOVA. D. The application of Nox1 siRNA and Nox4 siRNA together restored the DETA NONOate-induced inhibition of SMC migration in HG. *P<0.05 compared with cells treated with serum alone, N=8, one-way ANOVA.

Figure 4

Overexpression of Rac1 dominant-negative and p47^phox dominant-negative by adenovirus inhibit smooth muscle cell migration. A. Rac1DN and p47^phoxDN were overexpressed. B. Overexpression of Rac1 dominant-negative and p47^phox dominant-negative inhibited serum induced migration and restored the inhibition of migration by DETA NONOate. *P<0.05 compared with the lacZ group, N=9, one-way ANOVA.

Figure 5

Application of either H₂O₂ or DMNQ blocks the inhibition of SMC migration by NO. H₂O₂ or DMNQ were added 3 days prior to and after wounding the SMC layer. *P<0.05 compared with serum alone, N=5, one-way ANOVA.

Figure 6

Transfection of either Nox1 or Nox4 siRNA increases the reactive cysteine 674 thiol in SMC during high glucose exposure. A. Figure shows an example of three separate experiments, in which transfection of either Nox1 or Nox4 siRNA increased the ratio of bIAM labeled SERCA to total SERCA compared with control siRNA transfected SMC during HG treatment. B. Summary of the band density analysis from three separate experiments. *P<0.05 compared with control siRNA, one-way ANOVA.