Chemerin, a novel adipokine in the regulation of angiogenesis

Abstract

Context: Chemerin is a new adipokine associated with obesity and the metabolic syndrome. Gene expression levels of chemerin were elevated in the adipose depots of obese compared with lean animals and was markedly elevated during differentiation of fibroblasts into mature adipocytes.

Objective: The objective of the study was to identify factors that affect the regulation and potential function of chemerin using a genetics approach.

Design, setting, patients, and intervention: Plasma chemerin levels were measured in subjects from the San Antonio Family Heart Study, a large family-based genetic epidemiological study including 1354 Mexican-American individuals. Individuals were randomly sampled without regard to phenotype or disease status.

Main outcome measures: A genome-wide association analysis using 542,944 single-nucleotide polymorphisms in a subset of 523 of the same subjects was undertaken. The effect of chemerin on angiogenesis was measured using human endothelial cells and interstitial cells in coculture in a specially formulated medium.

Results: Serum chemerin levels were found to be highly heritable (h(2) = 0.25; P = 1.4 x 10(-9)). The single-nucleotide polymorphism showing strongest evidence of association (rs347344; P = 1.4 x 10(-6)) was located within the gene encoding epithelial growth factor-like repeats and discoidin I-like domains 3, which has a known role in angiogenesis. Functional angiogenesis assays in human endothelial cells confirmed that chemerin significantly mediated the formation of blood vessels to a similar extent as vascular endothelial growth factor.

Conclusion: Here we demonstrate for the first time that plasma chemerin levels are significantly heritable and identified a novel role for chemerin as a stimulator of angiogenesis.

Figure 1

Effect of recombinant chemerin concentration on blood vessel formation by endothelial cells in a coculture system. A coculture of endothelial cells and fibroblasts was used to study the tube formation by endothelial cells. The AngioKit was seeded with cells on d 0, and the optimized growth medium was changed on d 1, 3, 5, and 7. The cells were then fixed and stained for CD31 on d 9. Suramin (20 μm) and VEGF (2 ng/ml) were used as negative and positive controls, respectively. Recombinant human chemerin was added to each well at 0.1, 0.3, 1, 3, and 10 ng/ml. A, Control: cell medium only. B, Inhibition of tubule formation to control level by suramin (20 μm). C, VEGF (2 ng/ml) induced tubule formation as well as VEGF-induced increased branching of tubules and formation of in vitro capillaries. D–F, Coculture of endothelial cells and fibroblasts (AngioKit) after 9 d incubation in optimized growth media supplemented with 0.1, 1, and 10 ng/ml of recombinant human chemerin, respectively. The number of microtubules (G), tubule anastomoses (H), and microtubule length (I) were measured using an AngioSys image analysis system. Four images were captured per well from four independent quadrants. Experiments were run in triplicate, and data were expressed as the mean ± sem. *, P < 0.05, **, P < 0.01 compared with the vehicle control.

Figure 2

The effect of PD, a MEK1 inhibitor, on the angiogenic properties of chemerin in endothelial cells in a coculture system. The coculture of endothelial cells and fibroblasts (AngioKit) was used to study the formation of new blood vessels. The AngioKit was seeded with cells on d 0, and the medium was changed on d 1, 3, 5, and 7. Recombinant human chemerin was added at 3 and 10 ng/ml, respectively (in the presence or absence of 25 μm PD 98059). Cells were then fixed, and stained for CD31 on d 9. A, Control: cell medium only. B, Growth media with 0.1% DMSO. C, VEGF-positive control (2 ng/ ml). D, PD (25 μm). E, Recombinant human chemerin (3 ng/ml). F, Recombinant human chemerin (3 ng/ml) + PD (25 μm). The number of microtubule junctions (G), total number of microtubules (H), total microtubule length (I), and total tubule area (J) were measured using AngioSys image analysis system. Four images were captured per well from four independent quadrants. Experiments were run in triplicate, and data were expressed as the mean ± sem. *, P < 0.05, **, P < 0.01 compared with the vehicle control.