In the present study, we demonstrated that vaspin inhibits TNFα-induced NF-κB activation in vascular endothelial cells. Vaspin thereby inhibited NF-κB-dependent gene expression of the inflammatory and cell adhesion molecules ICAM-1, VCAM-1, E-selectin, and MCP-1. Furthermore, we investigated whether activation of AMPK by vaspin contributes to this inhibition of cytokine-induced NF-κB activation and expression of cell adhesion molecules. Transfection of AMPK α1 siRNA, which reduced AMPK expression by about 80% (Figure 5A), significantly attenuated vaspin-induced inhibition of TNFα-induced NF-κB activation and gene expression of various adhesion molecules in vascular endothelial cells. These data suggest that AMPK activation is responsible for vaspin-induced inhibition of NF-κB activation. Furthermore, this stimulatory effect of vaspin on the AMPK activation was independent of Akt and NO pathway, two representative signaling pathways linked to vaspin in vascular endothelial cells[18, 19, 26].
Although we did not perform a kinase assay of AMPK, vaspin likely activates AMPK. This is because the extent of AMPK phosphorylation at Thr172 strongly reflects its activity, and the consensus AMPK down-stream effector ACC was phosphorylated at Ser79 in vaspin-treated cells (Figure 1B and D). How vaspin activates AMPK in vascular endothelial cells remains to be elucidated; however, our data are in agreement with the effect of vaspin on AMPK activation in hepatocytes.
Vaspin was recently reported to prevent TNFα-induced ICAM-1 expression through its inhibitory effect on reactive oxygen species-dependent NF-κB activation in vascular smooth muscle cells. Furthermore, vaspin attenuates the induction of cytokines and vascular smooth muscle cell proliferation by high glucose levels. Vaspin elicits these effects by preventing the generation of reactive oxygen species and inhibiting NF-κB signaling. AMPK has pleiotrophic effects that may be anti-atherogenic, including the suppression of reactive oxygen species production induced by deleterious stimuli, such as hyperglycemia or high levels of free fatty acids[14, 31]. AMPK also has an anti-inflammatory effect through its inhibitory effects on free fatty acids or cytokine-induced NF-κB activation[14, 32]. Considering these effects, AMPK might be the upstream signaling molecule through which vaspin exerts its anti-atherogenic effect in vascular smooth muscle cells[29, 30]. Although further studies are required to investigate this hypothesis, our study is meaningful in that we firstly demonstrated the possible role of AMPK activation by vaspin in the aforementioned vaspin’s anti-inflammatory effect in vascular endothelial cells.
NF-κB is rapidly activated by various agents including inflammatory cytokines, such as TNFα, and is involved in a wide variety of biological phenomena including inflammatory and immune responses. Upon stimulation with various stimuli, the IκB kinase complex is activated and phosphorylates specific serine residues within IκB, which triggers the degradation of IκB and the subsequent nuclear translocation of p65 and p50. We demonstrated that vaspin inhibits the gene expression of proinflammatory and adhesion molecule genes by blocking TNFα-induced phosphorylation and subsequent degradation of IκBα (Figure 2D and E). These data suggest that vaspin might suppress TNFα-induced NF-κB activation before IκB phosphorylation. Although we did not examine the effect of vaspin on IκB kinase activity, the decreased activity of IκB kinase through vaspin-mediated AMPK activation might be the possible mechanism, considering the previous results which showed that AMPK can directly phosphorylate and attenuate the IκB kinase activity[24, 33, 34].
Several therapeutic approaches have been tried to ameliorate the endothelial injury by modulating the NF-κB-mediated signaling pathways such as knocking-down ultimate downstream effector and/or through the development of chemicals which help to reduce the NF-κB activity triggered by harmful stimuli. For example, knocking down the profilin-1, an actin-binding protein as well as the ultimate downstream effector in endothelial injury mediated by advanced glycation end products via NF-κB, attenuated the extent of endothelial abnormalities by reducing ICAM-1 and elevating NO levels. Regarding chemicals, propofol, a widely used intravenous anesthetic agent, has been demonstrated to decrease NF-κB activity, attenuated high glucose-induced endothelial adhesion molecules expression such as ICAM-1, VCAM-1 and E-selectin and mononuclear-endothelial adhesion. Vaspin can be considered as one of those molecules possessing anti-inflammatory effect via down-regulating the NF-κB pathway.
Recently, a cell-surface GRP78/voltage-dependent anion channel complex is considered as a potential receptor for vaspin in endothelial cells. Although the role of this GRP78/voltage-dependent anion channel complex in the activation of AMPK in vascular endothelial cells remains unclear, we could identify the role of GRP78 in the activation of AMPK pathway by vaspin in vascular endothelial cells by knocking down the expression of GRP78 using GRP78-specific siRNA (Online Additional file1: Figure S1). Further research is needed to elucidate how vaspin interacts with GRP78 and leads to the activation of AMPK in vascular endothelial cells.
AMPK is a heterotrimeric protein consisting of three subunits, α, β, and γ, each of which has at least two isoforms, which means that 12 complexes can form. These combinations generate AMPK complexes with different properties and tissue specificities. The α-subunit contains the catalytic site, whereas the β- and γ-subunits are important for maintaining the stability of the heterotrimer complex. The α1 isoform of AMPK is the predominant form in endothelial cells, although the α1 and α2 catalytic subunits are also expressed in these cells[39, 40]. This is why we knocked down the α1 isoform of AMPK using siRNA.
In the carotid arteries of a balloon injury rat model, treatment with an adenovirus vector expressing vaspin significantly suppresses the expression of MCP-1 and protects against vascular injuries, which is in agreement with the results of the current study. However, our study is the first to demonstrate the role of AMPK activation in the inhibitory effects of vaspin on cytokine-induced expression of proinflammatory molecules, including MCP-1, in vascular endothelial cells.
Emerging studies have revealed that visceral white adipocytes can act as an active endocrine tissue, secreting adipocytokines that play a key role in the relationship between obesity and the development of cardiovascular disease. There are suggested to be two types of adipocytokines, namely, ‘good adipocytokines’, of which adiponectin is probably the only well-established example, and ‘bad adipocytokines’, which may include inflammatory cytokines, such as TNFα, IL-6, MCP-1, and plasminogen activator inhibitor-1. Vaspin has been suggested to be a ‘good adipocytokine’[16, 42]. Vaspin has an anti-apoptotic effect in endothelial cells[19, 26, 43], and anti-inflammatory, and anti-migratory effects in vascular smooth muscle cells. Taking the results of these previous studies and the current study together, we propose that vaspin plays a protective role in the pathogenesis of atherosclerosis.
The serum level of vaspin in human varies according to the characteristics of the studied subjects[45–50]. In view of atherosclerosis, the vaspin levels varied among studies[50–52]. It has been suggested that an elevated level of vaspin is a compensatory factor in subjects with obesity or insulin resistance[17, 53, 54]. Considering the beneficial effect of vaspin on vascular cells demonstrated by previous studies[19, 26, 29, 43, 44], and this study, our data provides further evidence that vaspin is a compensatory factor.
It has been demonstrated that serum levels of vaspin measured with radioimmunoassay method varied from 0.2 to nearly 2 ng/ml in subjects with normal fasting plasma glucose and from 0.3 to nearly 3 ng/ml in subjects with type 2 diabetes. In our study, vaspin concentrations more than 25 ng/ml showed the anti-inflammatory (Figure 2C) and inhibitory effects against TNFα-induced monocyte adhesion to HAECs (Figure 4). These findings suggest that a higher concentration of vaspin might be needed to ameliorate the endothelial inflammatory status.
In conclusion, vaspin might attenuate cytokine-induced gene expression of adhesion molecule by inhibiting NF-κB following AMPK activation. These results provide a novel molecular mechanism underlying the anti-atherogenic effect of vaspin in endothelial cells. Further studies are needed to elucidate the specific mechanism by which vaspin activates AMPK in vascular endothelial cells.
Although vaspin, a recently identified adipocytokine in visceral adipose tissue of OLETF rat, has been suggested to have insulin sensitizing effect, its function in the body is largely unknown, especially in vascular cells. Previously, several studies had demonstrated that vapsin exerted anti-atherogenic effect on vascular cells. The present study provides a novel molecular mechanism that vaspin inhibits cytokine-induced expression of adhesion molecule genes by inhibiting NF-κB following AMPK activation. This study is thought to be meaningful in that it showed the novel function of vaspin in vascular cells and added another evidence supporting that vaspin acted as a compensatory factor.