In the present study, we have explored, for the first time, the protective effect of Zn supplementation on diabetes-induced pathogenic changes in the vessel, particularly in the aorta of genetic type 1 diabetic OVE26 mice. We found significant increases in aortic oxidative damage, inflammation, fibrosis and thickness in OVE26 mice, which was completely prevented by Zn treatment for 3 months. Mechanistic studies showed that the aortic protection of Zn treatment against diabetes-induced aortic pathogenesis was associated both with the up-regulation of Nrf2 protein expression and transcription, shown by the increased expression of Nrf2 down-stream gene NQO-1 at both mRNA and protein levels, and with the up-regulation of MT expression.
Zn plays important roles in the protection of vascular system from oxidative stress and damage. For instance, Zn deficiency in diabetic patients was found to associate with increased cardiovascular events , which may be related to the increased inflammatory response in system and vascular system [31–34]. Chronic inflammation plays a critical role for the development of various chronic pathogeneses [35–38]. The effects of chronic inflammation include induction of oxidative stress, apoptotic cell death, and abnormal cell proliferation, all which could contribute to the tissue structural and functional abnormalities [35–38]. In the present we demonstrated the induction of aortic inflammation, shown by increased expression of TNF-α, VACM-1 and PAI-1 in the aorta of DM, which was accompanied with increased aortic oxidative stress, apoptotic cell death, cell proliferation, and remodeling in DM group. All these pathogenic changes were prevented by Zn supplementation.
In contrast to Zn deficiency, Zn supplementation was found beneficial for the patients with various cardiovascular diseases [11, 14, 39–43]. Recently we also reported the aortic protection from diabetes-induced damage with sulforaphane in a type diabetic mouse model, in which, however, we have used streptozotocin (STZ) to induce diabetic mouse model , as used most frequently by others [45–48]. However, in STZ-induced diabetic animals STZ may have direct toxic effects on multiple organs . Compared to STZ-induced diabetic mice, OVE26 mice exhibit more characteristics of human diabetic nephropathy, showing the time-dependent proteinuria [28, 50, 51]. Therefore, here we used OVE26 and age-matched WT mice to demonstrate the aortic protection by Zn supplementation from diabetes-induced pathogenic damages, including oxidative damage, inflammation and fibrotic response.
In the present study we used both male and female mice to compare whether there is gender difference for the pathogenic changes in the aorta. No difference was found either for the pathogenic change in the aorta induced by diabetes or the preventive effect of Zn on diabetes-induced pathogenic changes in the aorta. Interestingly a previous study compared the impact of gender on cardiac contractile response in ventricular myocytes from wild-type FVB and OVE26 mice at young (2 month) and older (11 month) age . They found that OVE26 myocytes displayed reduced peak shortening (PS) and maximal velocity of shortening/relengthening (+/− dL/dt), and prolonged time-to-PS and time-to-90% relengthening, associated with higher resting intracellular Ca2+ levels and attenuated Ca(2+)-induced intracellular Ca2+ release compared with the FVB myocytes. Peak shortening and +/− dL/dt were smaller in female FVB groups when compared to the age-matched male counterparts. However, these gender differences were significant at 2 month old mice, but not at 11 month old mice. Therefore, mechanical differences existed between genders but were “cancelled off” by diabetic state. Nevertheless, a “female advantage” in ventricular function may still persist in young female diabetic subjects . Therefore, the age of OVE26 mice used here may explain the lack of gender impact on diabetes-induced pathogenic changes and its protection by Zn since these mouse ages are 6 months old.
One important mechanism by which Zn protects the aorta from diabetes may be related to the up-regulation of Nrf2 in the aorta. Recently the important protection by Nrf2 from diabetes in various organs, including the heart and kidney, has been extensively reported [17, 53, 54]. There were several reports to indicate the possible induction of Nrf2 and its down-stream antioxidant genes by Zn [14, 15, 55]. However, here we provided the first evidence to indicate the Zn protection against diabetes-induced pathogenic changes in the aorta of diabetic mice treated with Zn for 3 months, probably associated with the up-regulation of Nrf2 expression and function.
One of the novel findings in the present study is the difference for Nrf2 expression in the aorta of mice in response to diabetes and Zn. Nrf2 protein level (about 3 fold increase) is significantly lower than mRNA level (about 6 fold increase) in diabetes group, suggesting the increase of Nrf2 degradation, resulting in only about 3 fold increase in protein level. In contrast, Zn treatment did not increase Nrf2 mRNA level, but increased Nrf2 protein level to about 3 fold higher than control, suggesting Zn’s stabilization of Nrf2, which also explain why in DM/Zn group the Nrf2 expression is about 6 fold, since it is the combined outcome of the diabetic induction of Nrf2 mRNA and Zn stabilization of Nrf2 protein.
How Zn stabilizes Nrf2 protein level remains unclear, however, there was an interesting report that supports this stabilization theory . Chronic alcohol ingestion in rats decreases Zn levels and macrophage function in the alveolar space, which was accompanied with a decrease in Nrf2 nuclear binding capacity, i.e.: Nrf2 function decrease. When these rats were supplemented with Zn, Zn deficiency was corrected, resulting in a restoration of Nrf2 nuclear banding capacity along with a prevention of alcohol-induced macrophage function in the alveolar space of these rats . This study suggests the requirement of Zn for maintaining normal function of Nrf2 in certain conditions.
However, a question is why the increased pathologic change was still observed in the aorta of OVE26 diabetic mice that also showed a significant increase in aortic Nrf2 expression. We assumed that the expression of Nrf2 and its downstream antioxidants in the aortas of diabetic mice is an adaptive response to diabetes. Therefore, the increased level in DM group that is significantly less than that in DM/Zn group may be not enough to rescue all injuries. However, although this adaptive response is unable to completely prevent, it should still protect certain levels of pathogenic damage induced by diabetes; otherwise these pathogenic changes would be more severe and appear earlier.
In addition, Zn protection from diabetes-induced aortic injury may also include other possible mechanisms. One most likely alternative mechanism may be the induction of MT since MT’s anti-oxidative protection from various conditions, including diabetes, has been extensively reported, even in our own laboratory [18, 29, 56–59]. Here we provide the first evidence that Zn induced, and diabetes decreased, the MT mRNA expression in the aorta, but Zn treatment can preserve the MT mRNA expression to control level in the OVE26 mice. In contrast to aortic Nrf2 or NQO-1 expression that was also up-regulated in diabetic group, aortic MT expression was significantly decreased in DM group, but not in DM/Zn group.
Although there was no much information regarding the role of MT in vascular function under diabetic condition, a few studies related it under other conditions also support the important role of MT in maintenance of normal vascular structure and function [60, 61]. For instance, Zn protection from high-level glucose (HG), a diabetes feature on culture cardiomyocytes was observed recently . The authors demonstrated that extracellular Zn2+ reduced cardiomyocyte contractile function in both HG and control groups, but enhanced relaxation function significantly in the HG group compared to controls. Most notably, a reduction in diastolic sarcomere length with increasing pacing frequencies, i.e., incomplete relaxation, was more pronounced in the HG compared to controls, but was normalized with extracellular Zn2+ application, implicating that the detrimental effect of HG on cardiomyocyte Ca2+ regulation can be amelioration by Zn2+. Since MT was not measure in these cells, we do not know whether MT is involved in the beneficial effect of Zn. In another in vivo study, the effect of MT on collaterogenesis was demonstrated between mice with MT gene deletion and wild-type mice . They found that mice defect in MT gene expression have impaired collateral flow recovery after the induction of acute hind limb ischemia. They further demonstrated that endothelial cells, smooth muscle cells, and macrophages that are known to be involved in collateral remodeling were dysfunctional in MT gene deficient mice. All these studies imply that MT plays an important role in maintaining the normal structure and function of vascular cells and vessels. Zn beneficial effect on vascular cells and/or vessels maybe related to MT expression, which need to further study in the future.
A potential limitation of the present study is that the most results are based on the immunohistochemical staining and real-time PCR assays. For the protein expression, this study will be greatly strengthened if we could have enough tissues to perform Western blotting assay. This will be warranted in the further study.