E-selectin, constitutively expressed on endothelial cells, is a transmembrane glycoprotein which binds carbohydrate ligands on leukocytes, causing leukocyte rolling and facilitating endothelial adhesion and transmigration8
. TNF-alpha and other cytokines upregulate E-selectin gene expression8, 10, 19
and trigger enzymatic cleavage near the protein's membrane insertion point, with subsequent circulatory release of soluble E-selectin (sE-selectin)11, 12
. Levels of sE-selectin are thus thought to reflect systemic endothelial cell activation, though not necessarily endothelial cell dysfunction12
Previous studies have shown that serum E-selectin levels are elevated in obesity and decrease with weight loss14, 20, 21
. We confirmed this relationship between E-selectin and BMI in our cohort, and, interestingly, found that visceral adiposity may be more strongly associated with E-selectin than indices of total fat such as BMI or of subcutaneous fat. Both BMI and SAT were significant in univariate regression with E-selectin but were no longer significant in multivariate modeling including VAT. The importance of visceral fat as a predictor of E-selectin levels has also been suggested by a recent study of morbidly obese subjects20
, whereas, in a cohort of type 2 diabetics, BMI was a stronger predictor of E-selectin than VAT13
. We also demonstrate that E-selectin is associated with fasting insulin, glucose, triglyceride, and HDL levels, but these associations were no longer significant in a multivariate model including VAT. E-selectin has been associated with hyperinsulinemia22, 23
in previous studies, although these relationships may be mediated largely by excess body weight23
. Although other studies have demonstrated that E-selectin levels are higher in smoking24
, we did not find this association in our cohort.
We investigated the relationship between TNF-alpha and E-selectin and show in a human model of obesity that E-selectin is positively associated with sTNFR1and sTNFR2 levels. Soluble forms of TNFR1 and TNFR2 are produced by cleavage of the extracellular portion of the transmembrane receptors, which occurs both constitutively and in response to TNF-alpha and other cytokines25–27
. Thus, circulating levels of sTNFR1 and sTNFR2 reflect systemic inflammation and may be proportional to levels of membrane bound receptors. Studies have confirmed that activity of both TNFR1 and TNFR2 are necessary for the upregulation of E-selectin by TNF-alpha19
. Because causality can not be determined definitively in a cross-sectional study, we sought to further test the relationship between E-selectin and the TNF family by determining whether systemic treatment of obese subjects with a specific TNF-alpha antagonist, etanercept, would decrease E-selectin levels.
Treatment of obese subjects with etanercept significantly reduced E-selectin levels. The decrease in E-selectin was significantly associated with an increase in levels of TNF-alpha and sTNFR2. The directionality of change in TNF-alpha and sTNFR2 levels can be explained by drug-specific effects. Etanercept, a fusion protein between the recombinant human p75 TNF-alpha receptor 2 and the Fc fragment of human IgG1, binds TNF-alpha in the circulation, reducing its biological activity. Since etanercept sequesters TNF-alpha and prolongs its half life, circulating TNF-alpha levels rise with therapy. Moreover, as etanercept is detected in the assay for sTNFR2, measured sTNFR2 levels also rise with therapy. Thus, the significant association between decreases in E-selectin and increases in TNF-alpha and sTNFR2 supports the hypothesis that systemic TNF-alpha antagonism with etanercept influences E-selectin levels. In addition, decreases in E-selectin with etanercept were significantly associated with decreases in other serum inflammatory markers (as shown in ), suggesting a relationship between a reduction in E-selectin and other markers of subclinical inflammation in obesity. Of note, the reduction in E-selectin with etanercept occurred after only 4 weeks of treatment, suggesting rapid, acute effects of TNF-antagonism on E-selectin. These changes in E-selectin occurred in the absence of any changes in weight and VAT. Taken together our data suggest a schema whereby increased VAT may contribute to increased E-selectin through activation of the TNF family, but antagonism of TNF is sufficient to reduce E-selectin, independent of reduction in VAT. Thus unlike the studies of weight loss in which both weight and inflammation change simultaneously, the current model allows us to isolate the effects of TNF-alpha antagonism on E-selectin. While it is known that in patients with overt, inflammatory arthritides, TNF-alpha antagonism decreases E-selectin28, 29
, our data demonstrate a similar effect in subjects with no known cause for inflammation apart from obesity. The significance of visceral adipose tissue rather than subcutaneous or total adipose tissue in determining levels of E-selectin in our cohort may reflect disproportionate secretion of inflammatory cytokines by visceral fat compared with subcutaneous fat. In particular, visfatin, which upregulates TNF-alpha production, is predominantly produced by visceral adipose tissue and has been shown in vitro
to increase soluble E-selectin through activation of the NF-κB pathway30
. Further research is needed to clarify the specific mechanisms by which increased visceral fat may contribute to increased E-selectin.
The clinical significance of our findings depends on whether E-selectin simply serves as a marker of inflammation or whether it plays a role in human pathophysiology. A preponderance of evidence suggests the latter. The prevailing, inflammation-centric model of atherogenesis suggests that E-selectin and other leukocyte adhesion molecules help lure monocytes from the bloodstream to the tunica intima of affected vessels, where they acquire characteristics of tissue macrophages. These cells then internalize oxidized lipoprotein molecules, giving rise to foam cells that form the fatty core of an atheromatous plaque31, 32
. Intriguingly, a polymorphism of the E-selectin gene in humans is linked with a higher risk of early-onset atherosclerosis33
. Thus reductions in E-selectin via manipulation of the TNF-alpha axis in obesity may have clinical relevance. Given the potential side effect profile with etanercept, it is unlikely that this strategy will be pursued to reduce inflammation in obesity, but our data provide preliminary proof of the principle that such a strategy might be useful. Other strategies, including inhibition of nuclear factor kappa B with salsalate34, 35
, may be safer and equally effective, but have not been tested with respect to E-selectin.
Our study has a number of limitations. Although we performed both cross-sectional and interventional studies to assess the effects of TNF-alpha on E-selectin, we cannot be sure that the antagonism of TNF acts directly to downregulate E-selectin, or whether this effect is mediated through other pathways. Further studies are necessary to definitively conclude that TNF-alpha is directly causal in the pathway to increased E-selectin in obesity. Nonetheless, we see striking reductions in a number of inflammatory cytokines and endothelial markers, including E-selectin, after short-term antagonism of TNF, thus suggesting the potential utility of treating the subclinical inflammation of obesity in addition to the weight itself.