Angiotensin II Induces Oxidative Stress and Endothelial Dysfunction in Mouse Ophthalmic Arteries via Involvement of AT1 Receptors and NOX2

Figures

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  Figure 1. In vivo exposure to Ang II (0.5 mg/kg daily for 7 days) did not affect the response of ophthalmic arteries to the vasoconstrictor, U46619 (A), or to the endothelium-independent vasodilator, SNP (B), whereas it did cause a substantial degree of endothelial dysfunction (impaired acetylcholine response, (C)). Values are expressed as mean ± SE (n = 8 per group; * p < 0.05, Ang II 0.5 mg/kg/d versus vehicle control). In vitro exposure of ophthalmic arteries to different concentrations of Ang II also had no effect on the response to U46619 (D) or SNP (E), but markedly blunted reactivity to acetylcholine at 10−7 M of Ang II (F), indicative of endothelial dysfunction. Values are expressed as mean ± SE (n = 8 per group; * p < 0.05, Ang 10−7 M versus vehicle control).

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  Figure 2. The AT1R blocker, candesartan, prevented Ang II-induced endothelial dysfunction (A) (n = 8 per group; ** p < 0.01, Ang II 10−7 M versus vehicle; # p < 0.01, Ang II 10−7 M versus vehicle + candesartan; * p < 0.05, Ang II 10−7 M versus Ang II 10−7 M + candesartan). The AT2R blocker, PD 123319 ditrifluoroacetate, had no effect on Ang II-induced endothelial dysfunction (B) (n = 8 per group; *** p < 0.001, Ang II 10−7 M + PD 123319 ditrifluoroacetate versus vehicle; ** p < 0.01, Ang II 10−7 M versus vehicle; # p < 0.01, vehicle + PD 123319 ditrifluoroacetate vs. Ang II 10−7 M + PD 123319 ditrifluoroacetate; * p < 0.05, vehicle + PD 123319 ditrifluoroacetate vs. Ang II 10−7 M). Data are expressed as mean ± SE.

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  Figure 3. While Ang II 10−7 M reduced endothelium-dependent vasodilation (reduced acetylcholine responses), the ROS scavenger Tiron 10−3 M retained acetylcholine-induced vascular responses in Ang II 10−7 M-incubated ophthalmic arteries (A). Ang II and Tiron had no effect on the SNP response (endothelium-independent vasodilation) (B). Data are expressed as mean ± SE (n = 8 per group; ** p < 0.01, Ang II 10−7 M versus vehicle + Tiron; + p < 0.01, Ang II 10−7 M vs. Ang II 10−7 M + Tiron; * p < 0.05, Ang II 10−7 M versus vehicle). Ang II 10−7 M increased fluorescent intensity to DHE in ophthalmic artery cross-sections, indicative of elevated ROS levels, whereas Tiron prevented an Ang II-induced increase in DHE staining intensity (C,D). Data are expressed as mean ± SE (n = 8 per group; *** p < 0.001; ** p < 0.01; * p < 0.05). Scale bar = 50 µm.

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  Figure 4. Incubation with Ang II (10−7 M) induced marked expression of NOX2 mRNA in isolated ophthalmic arteries of C57Bl/6J mice. In contrast, NOX1 mRNA was undetectable, and NOX4 and XDH/XO mRNA only insignificantly increased in Ang II-incubated arteries compared to vehicle-incubated arteries (A) (n = 6 per group, ** p < 0.01 versus vehicle control). While immunoreactivity to NOX2 was only weak in vehicle-incubated ophthalmic arteries from C57Bl/6J mice (B,C), much stronger immunoreactivity to NOX2 was seen in the vascular wall of arteries incubated with Ang II 10−7 M (B,C) (n = 8 per group, *** p < 0.001, Ang 10−7 M versus vehicle control). Vasoconstrictor responses to U46619 (D), endothelium-independent vasodilation to SNP (E), and endothelium-dependent vasodilation to acetylcholine (F) were not reduced in isolated ophthalmic arteries from NOX2–/– mice exposed to Ang II 10−7 M compared to vehicle, suggesting that Ang II failed to induce endothelial dysfunction when NOX2 was not present. The curves with dashed lines were reproduced from Figure 1D–F for the direct comparison with the respective control condition. Data are expressed as mean ± SE (n = 8 per group; ** p < 0.01, NOX2+/+ +…

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  Figure 5. The non-isoform-selective COX blocker, indomethacin (Indo), had no effect on the acetylcholine-induced (endothelium-dependent) vasodilation in the vehicle-incubated and Ang II 10−7 M-incubated ophthalmic arteries from wild-type mice (C57BL/6J) (A). In contrast, the non-isoform-selective NOS inhibitor, L-NAME, markedly reduced the endothelium-dependent vasodilation in the vehicle-incubated arteries, but only had a negligible effect in Ang II-incubated arteries (B) (* p < 0.05, vehicle + L-NAME versus vehicle). Also, the combined blockade of COX and NOS significantly reduced endothelium-dependent vasodilation in vehicle-incubated but not in Ang II-incubated ophthalmic arteries (C) (** p < 0.01, vehicle + Indo + L-NAME versus vehicle). KCl (30 mM), which prevents hyperpolarization, virtually abolished endothelium-dependent vasodilation in both vehicle- and Ang II-incubated vessels, in which COX and NOS had been blocked, indicative of substantial EDHF involvement (D) (*** p < 0.001, vehicle + Indo + L-NAME + KCl versus vehicle + Indo + L-NAME and Ang II 10−7 M + Indo + L-NAME + KCl versus Ang II 10−7 M + Indo + L-NAME). Inhibition of CYP450 by 17-ODYA moderately blunted…

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  Figure 6. Proposed hypothetical signaling pathways affected by Ang II in the mouse ophthalmic artery endothelium. Activation of the angiotensin receptor type 1 (AT1R) by angiotensin II activates NADPH oxidase 2 (NOX2) in the cytoplasmic membrane via diacylglycerol (DAG), protein kinase C (PKC), and phosphorylation of the NADPH oxidase subunit p47phox to P-p47phox. Moreover, NOX2-derived ROS were shown to open mitochondrial KATP channels (mtKATP) in the mitochondrial membrane subsequently causing the opening of permeability transition pores (mPTP) providing an amplification mechanism for Ang II-induced oxidative stress. The ROS signaling mechanism is summarized from previous publications [9,56,64]. Based on our findings, NOX2-derived reactive oxygen species (ROS) inactivate endothelial nitric oxide synthase (eNOS), which leads to diminished nitric oxide (NO) production. Moreover, impairment of 12/15-lipoxygenase (12/15-LOX) by ROS may blunt the generation of 12-hydroxyeicosatetraenoic acid (12-HETE), which is the predominant HETE regioisomer in the mouse and which appears to act as an endothelium-derived hyperpolarizing factor (EDHF) [65]. In contrast, cytochrome P450 oxygenases (CYP450),…