Air pollution exposure potentiates hypertension through reactive oxygen species-mediated activation of Rho/ROCK

Abstract

Objective: Fine particulate matter <2.5 microm (PM(2.5)) has been implicated in vasoconstriction and potentiation of hypertension in humans. We investigated the effects of short-term exposure to PM(2.5) in the angiotensin II (AII) infusion model.

Methods and results: Sprague-Dawley rats were exposed to PM(2.5) or filtered air (FA) for 10 weeks. At week 9, minipumps containing AII were implanted and the responses studied over a week. Mean concentration of PM(2.5) inside the chamber was 79.1+/-7.4 microg/m(3). After AII infusion, mean arterial pressure was significantly higher in PM(2.5)-AII versus FA-AII group. Aortic vasoconstriction to phenylephrine was potentiated with exaggerated relaxation to the Rho-kinase (ROCK) inhibitor Y-27632 and increase in ROCK-1 mRNA levels in the PM(2.5)-AII group. Superoxide (O(2).(-)) production in aorta was increased in the PM(2.5)-AII compared to the FA group, inhibitable by apocynin and L-NAME with coordinate upregulation of NAD(P)H oxidase subunits p22(phox) and p47(phox) and depletion of tetrahydrobiopterin. In vitro exposure to ultrafine particles (UFP) and PM(2.5) was associated with an increase in ROCK activity, phosphorylation of myosin light chain, and myosin phosphatase target subunit (MYPT1). Pretreatment with the nonspecific antioxidant N-acetylcysteine and the Rho kinase inhibitors (Fasudil and Y-27632) prevented MLC and MYPT-1 phosphorylation by UFP suggesting a O(2)(.-)-mediated mechanism for PM(2.5) and UFP effects.

Conclusions: Short-term air pollution exaggerates hypertension through O(2)(.-)-mediated upregulation of the Rho/ROCK pathway.

Figure 1

A, Twenty-four hour mean arterial blood pressure in the aorta (MAP) at baseline and after 9 weeks of PM_2.5 exposure (Pre-AII) in SD rats (n = 6). There were no significant changes in mean arterial pressure following 9 weeks of PM_2.5 exposure alone. B, MAP change in SD rats exposed to PM_2.5-AII or FA-AII after the implant of AII osmotic minipumps (n = 6).

Figure 2

Aortic vasomotor responses in SD rats exposed to PM_2.5-AII or FA-AII. Contraction of aortic rings in response to vasoconstrictor phenylephrine (A), and relaxation of aortic rings in response to endothelium dependent vasodilator acetylcholine (B) or ROCK inhibitor Y-27632 (C) (n = 6). *P < 0.05 vs. FA-AII for peak constriction or dilation, †P < 0.05 vs. FA-AII for EC₅₀ or ED₅₀.

Figure 3

Expression of NAD(P)H oxidase subunits, RhoA, and ROCKs in aortic tissue of rats exposed to FA-AII or PM_2.5-AII. A and B, The mRNA expression of NAD(P)H oxidase subunits p22^phox, p47^phox, p67^phox (A), and Nox1, Nox2, Nox4 (B) in aortic tissue. C and D, The mRNA expression of ROCK-1 and ROCK-2 (C), and RhoA (D) in aortic tissues. The mRNA levels were determined by real-time quantitative PCR, and normalized to that of β-actin mRNA. E, The expression of RhoA protein in aortic tissue. Values are expressed as mean ± SE (n= 6). *P < 0.05 vs. FA-AII.

Figure 4

PM exposure induces MLC activation through RhoA/ROCKmediated pathways in cultured rat aortic smooth muscle cells. For all experiments, cells were serum starved and exposed to UFP or PM_2.5 for the indicated time or dose. A. The level of “active” GTP-RhoA in cell lysates detected with a specific anti-RhoA antibody. Values are expressed as mean ± SE (n= 3) *P < 0.05 vs. 0 min. B. Time course of MLC phosphorylation with UFP. Cells were exposed to 10 µg/ml of UFP for the indicated time and MLC phosphorylation was determined by immunoblotting. C. Comparison of UFP with PM_2.5 on MLC phosphorylation. Cells were exposed to 10 µg/ml of UFP or PM_2.5, or to 100 nM of AII (positive control) for the indicated time. MLC phosphorylation was determined by immunoblotting. D. Effect of UFP and PM_2.5 on MLC phosphorylation contrasted by dose. 10 or 50 µg/ml of UFP or PM_2.5 were used for 5 min with MLC phosphorylation determined by immunoblotting. E. Effect of UFP compared with AII on phosphorylation of large sub-unit of myosin phosphatase-MYPT1. Cells were treated with 10 µg/ml of UFP and/or 100 nmol/L of AII for 3 min, and the phosphorylation of MYPT1 and MLC were detected. F. PM effects on MLC phosphorylation are mediated by ROS and Rho/ROCK pathways. Cells were exposed to 10 µg/ml of UFP for 3 min with or without pretreatment for 20 minutes with the thiol anti-oxidant N-acetylcysteine (NAC, 5 mM), Y-27632 (10 µM) and fasudil (10 µM). All data shown are representative of 3–4 independent experiments.