Previous studies have implicated a protective role for intracellular SOD1 as well as extracellular SOD3 in limiting endothelial dysfunction produced by AngII.4, 5, 24
In the present study, we demonstrate that Ang II infusion increases Atox1 expression in aortas of wild-type mice. Activity and expression of SOD3, but not those of SOD1, are decreased in Atox1−/−
mice treated with Ang II. This leads to exaggerated Ang II-induced vascular O2•−
production, thereby promoting Ang II-induced endothelial dysfunction and vasoconstriction, which in turn augments systolic blood pressure in Atox1−/−
mice. These changes induced by Atox1 deficiency are rescued by adding SOD mimetic tempol. Thus, Atox1 functions to prevent Ang II-induced endothelial dysfunction and hypertension by reducing O2•−
levels via increasing vascular SOD3 expression and activity. Moreover, Atox1 regulates copper levels in the vasculature during Ang II-induced hypertension.
Impairment of endothelium-dependent ACh-induced relaxation in mesenteric arteries from Ang II-infused Atox1−/−
mice is likely due to a decrease in endothelial NO.
bioavailability, because endothelium-independent vasodilation is not affected by Ang II in Atox1−/−
mice. There is significant reduction in vascular reactivity in vehicle-infused Atox1−/−
mice, which may be in part due to the slight, but not significant, increase in O2•−
levels in these mice at baseline. However, Ang II-induced hypertension, but not basal blood pressure, is significantly altered in Atox1−/−
mice. In addition, it has been reported that Ang II increases vasoconstriction partially through O2•−
-induced inactivation of vasorelaxant NO1
, although this response depends on vascular-bed and genetic background.25, 26
In the present study, we found that contractions to the Ang II, but not KCl, are significantly increased in mesenteric arteries of the Atox1−/−
mice compared with WT mice. This is inhibited by SOD mimetic tempol. Thus, the loss of NO bioavailability induced by decrease in SOD3 activity and subsequent increase in O2•−
levels in Atox1 deficient arteries may contribute not only to impaired endothelium-dependent vasodilation but also to enhanced Ang II-induced vasoconstriction. This in turn likely increases systemic vascular resistance in Atox1−/−
mice during Ang II infusion and, thus, augments the hypertensive response observed in these animals. Of note, it has been shown that endothelium-dependent vasorelaxation in mesenteric arteries is dependent on not only NO but also epoxyeicosatrienoic acids (EETs) and H2
, which have been proposed as endothelium-dependent hyperpolarizing factor (EDHF)27
. Thus, Atox-1−/−
mice have increased vascular O2•−
which would scavenge NO, but also may have reduced vascular H2
to inhibit action of EDHF. This point requires further investigation in future study.
We found that the levels of total SOD activity were similar between WT and Atox1−/−
mice and were not changed by Ang II infusion. This result may reflect the fact that SOD1 activity, which consists of more than half of total SOD activity, is not dependent on Atox1. Nevertheless, the dramatic decrease in SOD3 activity by Atox1 deficiency likely has a major impact on increasing O2•−
levels in the interstitial space, where this enzyme is localized. This will efficiently promote oxidative inactivation of NO which traverses from the endothelial cells to VSMCs, thereby preventing endothelium-dependent vasorelaxation, which in turn increases blood pressure in Atox1−/−
mice, as reported for SOD3−/−
Of note, endothelial dysfunction in the coronary circulation of humans has profound prognostic implications in that it predicts adverse cardiovascular events and long-term outcome.28
The R213G polymorphism in the SOD3 gene, which reduces binding to endothelial surface and increases serum SOD3 levels,3
has been linked to an increase in cardiovascular risk.29
Thus, protective role of Atox1 in endothelial function through SOD3 would have potential clinical impact.
In this study, SOD3 protein and mRNA expression as well as nuclear Atox1 expression in blood vessels are increased by Ang II infusion, which are abolished in Atox1−/−
mice. Consistent with our results, Hamza and Gitlin originally showed that Atox1 is localized in the nucleus in cultured cells30
; however, functional significance of nuclear Atox1 was not addressed. Using promoter analysis and DNA pull-down assay in cultured VSMCs, we demonstrate that Ang II stimulation promotes translocation of Atox1 from the cytosol to the nucleus as well as Atox1 binding to the Atox1 response element in SOD3 promoter. This observation is consistent with our earlier study that Atox1 serves as a transcription factor for copper-induced increase in SOD3 and cyclin D1 expression in fibroblasts.9, 12
The present study also found that expression of other copper containing enzymes including lysyl oxidase, ceruloplasmin or SOD1 was not changed in Atox1−/−
arteries as compared to WT with or without Ang II infusion. This is consistent with the fact that SOD3, but not lysyl oxidase, ceruloplasmin or SOD1, has Atox1-responsive elements in its promoters. Thus, current study provides the first evidence that “agonist-induced” transcription factor function of Atox1 for SOD3 plays a potentially important role in Ang II-induced upregulation of SOD3 expression in blood vessels, and thereby modulating O2•−
levels and hypertensive responses induced by Ang II ().
Proposed model for protective role of Atox1 in Ang II-induced hypertension through regulating SOD3 expression and activity
Copper chaperone function of Atox1 to deliver copper to SOD3 in Ang II-treated VSMCs is also demonstrated in this study. We found that Ang II promotes Atox1 binding to the copper exporter ATP7A which obtains copper from Atox1. It also stimulates ATP7A translocation from TGN to plasma membrane where it colocalizes with SOD3. Consistent with our results, Atox1 is shown to deliver copper to secretory copper enzymes via interaction with ATP7A in yeast.21, 22
Furthermore, ATP7A is reported to deliver copper to the secretory copper enzymes in the postGolgi vesicles rather than in TGN where copper loading normally takes place.31, 32
We previously reported that Ang II infusion-induced increase in blood pressure and vascular O2•−
production are augmented in ATP7A mutant mice due to decrease in SOD3 activity.33
In line with this, the present study provides the additional new evidence that Ang II promotes copper chaperone function of Atox1 by facilitating formation of Atox1/ATP7A/SOD3 complex, thereby increasing specific SOD3 activity ().
The association of copper metabolism with hypertension has been implicated.13
However, there is no information regarding vascular copper levels in hypertension, or involvement of Atox1 in this response. The present study shows that Ang II treatment significantly decreases vascular copper levels as assessed by ICP-MS and SXFM analysis, which is inhibited in Atox1−/−
mice. This might in part reflect the Ang II-stimulated secretion of copper-loaded SOD3 to the circulation via the Atox1-ATP7A pathway, despite the majority of SOD3 will bind to the extracellular matrix. In addition, Ang II-induced decrease in vascular copper levels may be also due to secretion of secretory copper containing proteins, or excess copper to protect cells from its toxicity. This is consistent with previous study that levels of copper in some tissues such as liver and kidney are significantly lower in hypertensive rodents.14
The physiological consequence of copper export via ATP7A is placental copper transport to the developing fetus during pregnancy or to provide copper as part of a neuronal protective mechanism.34
Thus, the present study provide the first evidence that vascular copper levels are altered during Ang II-induced hypertension, which is at least in part regulated by Atox1. Detailed analysis of molecular mechanism and functional significance of altered copper levels in hypertension requires future investigation.
Previous studies have shown that SOD3−/−
mice have normal blood pressure at baseline but Ang II-induced increase in blood pressure is augmented in these mice in a time dependent manner with a peak at day 7, which remained elevated at least until day 14.5
Consistent with this, the present study found that Atox1−/−
mice showed similar time course of exaggerated hypertensive response to Ang II, as reported for SOD3−/−
mice. It has been reported that deletion of SOD3 in the circumventricular organ (CVO) in the brain increases blood pressure in the basal state and after Ang II infusion, in part by modulating sympathetic outflow.35
Intriguingly, Atox1 expression is observed in several regions of brain including choroid plexus which belongs to CVO, where ATP7A is also expressed.36
Thus, it is conceivable that Atox1 may be involved in regulation of blood pressure, either by regulation of SOD3 in CVO or by other secretory copper enzymes in the brain. Atox1 is also expressed in the kidney, including glomeruli in both the juxtramedullary and cortical nephrons and medulla associated with the loops of Henle,37
which regulates oxidative stress-dependent hypertension.38
In particular, O2•−
production in the brain is required for the genesis of hypertension.35, 39
In current study, we used non-invasive tail cuff method to measure blood pressure. However, it does not give the information, such as 24-hour blood pressure and blood pressure variability. Thus, Atox1-SOD3-mediated regulation of Ang II-induced hypertension should be further confirmed using telemetry system. Furthermore, investigating role of Atox1 in other oxidative stress-dependent pathophysiologies such as diabetes mellitus, obesity, and atherosclerosis in which blood pressure is not consistently increased38
is the subject of future studies.
The present study provides compelling evidence that Atox1 plays an important role in regulating vascular function and hypertension induced by Ang II in vivo by decreasing extracellular O2•− and increasing bioavailability of NO through copper chaperone and transcription factor function for SOD3 in blood vessels (). Atox1 is also involved in regulating copper homeostasis during Ang II induced hypertension. Our findings provide novel insight into Atox1 as a potential therapeutic target for treatment of hypertension and various other oxidative stress-dependent cardiovascular diseases.