Figure 1: Expression of TSG-6 in Human Vascular Cells and its Effects on Inflammatory Response and Proliferation in Human ECs

(A) Aliquots of 40 μg of cellular protein from human monocytes, HMDMs, HASMCs, and HUVECs were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to immunoblotting analysis for human TSG-6 and β-actin. Immunoblots are representative of 2 independent experiments. (B) Proliferation of EA.hy926 ECs was determined by WST-8 assay after 48-h incubation in DMEM containing 2 concentrations of d-glucose (Glc) (1.0 or 4.5 mg/ml) with the indicated concentrations of TSG-6. EA.hy926 ECs cultured in 4.5 mg/ml d-glucose–conditioned medium were stained by hematoxylin eosin. Glc 1.0 mg/ml (blue): n = 4; *p < 0.005; †p < 0.0005; ‡p < 0.0001 vs. 0 ng/ml of TSG-6. Glc 4.5 mg/ml (red): n = 5; #p < 0.01 vs. 0 ng/ml of TSG-6. (C) HUVECs were pre-treated with or without TSG-6 (300 ng/ml) for 30 min and then incubated with or without LPS (1 μg/ml) for 2 h. The levels of IL-6, TNF-α, MCP-1, ICAM-1, VCAM-1, E-selectin, and GADPH mRNA expression were measured by reverse transcription polymerase chain reaction. The graph shows the densitometric data of each molecule after normalization relative to GADPH. n = 3 to 4; §p = 0.028; ¶p < 0.005 vs. control; @p < 0.01; &p = 0.028 vs. LPS. All values are presented as mean ± SEM. EC = endothelial cells; GAPDH = glyceraldehyde-3-dehydrogenase; Glc = d-glucose; HASMC = human aortic smooth muscle cell; HMDM = human monocyte-derived macrophage; HUVEC = human umbilical vein endothelial cell; ICAM = intercellular adhesion molecule; IL = interleukin; LPS = lipopolysaccharide; MCP = monocyte chemotactic protein; mRNA = messenger ribonucleic acid; TNF = tumor necrosis factor; TSG = tumor necrosis factor–stimulated gene; VCAM = vascular adhesion molecule.