Ambient ultrafine particles reduce endothelial nitric oxide production via S-glutathionylation of eNOS

Abstract

Exposure to airborne particulate pollutants is intimately linked to vascular oxidative stress and inflammatory responses with clinical relevance to atherosclerosis. Particulate matter (PM) has been reported to induce endothelial dysfunction and atherosclerosis. Here, we tested whether ambient ultrafine particles (UFP, diameter <200 nm) modulate eNOS activity in terms of nitric oxide (NO) production via protein S-glutathionylation. Treatment of human aortic endothelial cells (HAEC) with UFP significantly reduced NO production. UFP-mediated reduction in NO production was restored in the presence of JNK inhibitor (SP600125), NADPH oxidase inhibitor (Apocynin), anti-oxidant (N-acetyl cysteine), and superoxide dismutase mimetics (Tempol and MnTMPyP). UFP exposure increased the GSSG/GSH ratio and eNOS S-glutathionylation, whereas over-expression of Glutaredoxin-1 (to inhibit S-glutathionylation) restored UFP-mediated reduction in NO production by nearly 80%. Thus, our findings suggest that eNOS S-glutathionylation is a potential mechanism underlying ambient UFP-induced reduction of NO production.

Keywords: Air pollution; Endothelial dysfunction; Oxidative stress; S-glutathionylation; Ultrafine particles/UFP; eNOS.

Fig. 1. UFP-induced oxidative stress reduced NO production

(A) HAEC were treated with an incremental concentration of UFP for 6 hours. NO production was assessed in terms of Nitrite /Nitrate concentration in cell lysate. (B) and (C) HAEC were pre-treated with 2μM of SP600125 (SP) or 200 μM of Apocynin (APO), and with SOD1 mimetics, Tempol, at 200μM, SOD2 mimetic, MnTMPyP, at 2μg/mL, or an antioxidant N-acetyl Cysteine (NAC, 1mM) for 30 minutes, followed by co-treatment with or without 50μg/mL of UFP for 6 hours. Inhibition of JNK or NADPH oxidase in (B) as well as mitigation of ROS by antioxidants in (C) restored UFP-induced inhibition on NO production (n=3).

Fig. 2. UFP increased protein S-glutathionylation in HAEC

(A) HAEC were treated with or without 50μg/mL of UFP for 6 hours, cellular levels of GSH and GSSG were measured as described in Methods. UFP decreased GSH level, but increased GSSG level. (n=4) (B) HAEC were treated with or without 50μg/mL of UFP for 6 hours and protein lysates were collected. Western blots with antibody against GSH revealed an increase in a dominant band to S-Glutathionylated Actin. The western blot with anti-Tubulin was performed as loading reference. (C) Sections of endocardium from LDLR-null mice exposed to filtered air (FA) or UFP for 10 weeks were stained with anti-GSH antibody for visualization of protein S-glutathionylation. UFP exposure led to a prominent staining in the endothelium of endocardium (Arrow).

Fig. 3. UFP stimulated eNOS S-glutathionylation

HAEC were treated with 50μg/mL of UFP or 25μM BCNU (a positive control) for 6 hours. eNOS S-glutathionylation was measured as described in Methods. UFP significantly increased eNOS S-glutathionylation (n=3, p < 0.01).

Fig. 4. S-glutathionylation mediated UFP-reduced NO Production

(A) HAEC were infected with control (Adv-LacZ) or Glutaredoxin-1(Adv-Grx-1) adenoviruses at different multiple of infection (MOI) overnight. Grx-1 expression was assessed by western blot. (B) HAEC were infected with Adv-LacZ or Adv-Grx-1 at MOI of 1:100 overnight. The cells were then treated with or without 50μg/mL of UFP for 6 hours. NO production was measured. Over-expression of Grx-1 attenuated UFP-mediated inhibition in NO production (n=6).