This study has demonstrated that daily injections of OCN produced significant effects on glucose and lipid metabolism, as well as improving insulin sensitivity, in ApoE-KO mice, all of which represent risk factors of cardiovascular disease. Moreover, vascular EDR was significantly improved in thoracic aorta specimens from the OCN-treated ApoE-KO mice fed a high fat diet. Following incubation of the HUVECs or thoracic aortic strips with OCN, eNOS phosphorylation was significantly increased and HFD-related impairment of EDR was attenuated. It was determined that the protective effect of OCN was mediated, at least in part, by the activation of PI3K/Akt signaling pathway, which was consistent with the research of Jung et al.. These results suggest that OCN plays an important role in modulating endothelial function in vivo.
Previous studies have shown that, in accord with the present study, the osteoblast-derived protein, OCN, affects lipid and glucose metabolic regulation in mice. In the current study of ApoE-KO mice, the daily injections of OCN induced decreases in FBG and serum level of lipids, and an increase in insulin secretion regardless of diet composition. Although the OCN injections had no significant effects on glucose tolerance and insulin sensitivity in the ApoE-KO mice on a chow diet, they did improve glucose tolerance and insulin sensitivity in ApoE-KO mice on a HFD. This suggests that intermittent injections of OCN have a more profound effect in mice with already altered insulin sensitivity. Systemic metabolic abnormalities, such as dyslipidemia, hyperglycemia and insulin resistance, are important risk factors of cardiovascular disease[20, 21]. Hypercholesterolemia is one of the characteristics of ApoE-KO mice. Substantial clinical and experimental evidence has suggested that both hyperglycemia and dyslipidemia contribute to endothelial cell dysfunction. Hyperglycemia causes an accelerated formation of advanced glycation end-products and mitochondrial overproduction of reactive oxygen species. Dyslipidemia also strongly and directly enhances monocyte adhesion to endothelium. Both alterations can result in vascular injury and endothelial damage. Given that OCN can ameliorate dyslipidemia and impaired glucose metabolism, it may well constitute a protective factor for vascular disease. TNF-α, IL-1α and IL-12 are known as pathogenic factors during the development of atherosclerosis. Our results suggested that OCN may also play a role in alleviating chronic inflammation in ApoE-KO mice. In addition, several clinical studies have reported a significantly inverse correlation between OCN and blood pressure[23, 24]. In the present study, a significant vascular protective effect of OCN, through its lowering of mean BP and diastolic BP, was found in ApoE-KO mice fed the HFD.
The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several vasodilators, including NO and endothelium-derived hyperpolarizing factor (EDHF). In mouse aorta, NO is the sole endothelial-derived mediator. Substantial clinical and experimental evidence suggests that endothelial dysfunction is an early marker for the initiation and progression of atherosclerosis. ApoE-KO mice, as in the present study, are one of the most widely used animal models of atherosclerosis[28–30]. Endothelial dysfunction has been demonstrated in the aorta of ApoE-KO mice fed a western diet and is characterized by impaired ACh-induced endothelium-mediated aortic vasodilation. In accord with previous studies, our research found a reduced EDR to ACh in the aorta of ApoE-KO mice fed with HFD; this result serves as an important indicator of vascular dysfunction. Furthermore, an increased EDR to ACh was demonstrated in the OCN-treated HFD group. By contrast, the phenomenon was not observed in mice fed with chow diet. Previous studies suggested that the expression of total Akt significantly increased in HFD status, which may indicate a compensatory or adaptive mechanism in obese mice[31–33]. This might explain why OCN had a more significant effect on EDR in mice fed with HFD through the regulation of the Akt/eNOS-dependent pathway. Application of L-NAME abolished EDR in all of the ApoE-KO mice, regardless of diet composition, similar to the findings of other researchers. However, OCN did not appear to affect the ability of VSMCs to respond to NO. Thus, OCN may play a positive role in endothelial vasodilatory function and this beneficial effect may result from, at least partially, its favorable modulation of glucose and lipid metabolism by way of its protective effect against endothelial dysfunction otherwise induced by glucose and lipid metabolism disorders.
This study also sought to determine the possible mechanism that underlies the relationship between OCN and vascular EDR. Altered eNOS-NO signaling is a common feature observed in animal models exhibiting impaired endothelial function[35–38]. Phosphorylation at Ser1177 is necessary for the maximal activation of eNOS and results in optimal NO production. In the present study, expression of P-eNOS and total eNOS was increased in descending thoracic aortic strips of OCN-treated ApoE-KO mice fed with HFD, compared with the vehicle-treated ApoE-KO mice fed with HFD. In addition, similar results were found in OCN-treated HUVECs, and this in vitro finding agreed with those of a previous study by this group. Collectively, these results suggest that eNOS-NO signaling may contribute to the OCN-mediated modulation of vasorelaxation in ApoE-KO mice.
In the endothelium, PI3K/Akt signaling mostly acts as a positive regulator of endothelial NO synthase, which generates NO through the NADPH-dependent oxidation of L-arginine[40, 41]. Regarding the regulation of eNOS by PI3K/Akt signaling, it has previously been shown that activated Akt phosphorylates eNOS on Ser1177, enhancing both basal and stimulated eNOS enzyme activity, and thereby NO release[42, 43]. In contrast, loss of the Akt1 subtype in the vessel wall is associated with reduced eNOS phosphorylation[44, 45]. In the present study, phosphorylation of both Akt and eNOS was significantly increased in HUVECs in response to OCN treatment. Furthermore, a beneficial effect of OCN was observed using an ex vivo organ culture of isolated mouse aortic strips, which was attenuated upon coincubation with a PI3K or Akt inhibitor. These results suggest that OCN may protect vascular endothelial cell from impairment, in part, by activating the PI3K/Akt signaling pathway.