Effect of leptin on lymphatic endothelial cell tube formation and proliferation.

(A) Representative phase contrast microscopy images showing tube formation in HDLECs treated with leptin (10, 100 ng/ml) for 17 h with or without anti-leptin antibody (10 μg/ml). Scale bar: 50 μm. (B) Histograms represent total tube length in HDLECs exposed to leptin (10, 100 ng/ml) for 17 h with or without 1 or 10 μg/ml anti-leptin antibody. Total tube length and mean length in each condition were measured using Image J software. Results are expressed as means ± SEM of four independent experiments; *p < 0.05, **p < 0.01, t-test. n.s.: not significant. (C) WST-8 assay showing proliferation of HDLECs treated with leptin (10, 100 ng/ml) for 17 h with or without 1 or 10 μg/ml anti-leptin antibody. Histograms represent absorbance at 450 nm. Results are expressed as means ± SEM of four independent experiments; *p < 0.05, t-test. n.s.: not significant. (A-C) Anti-leptin antibody was added 30 min prior to leptin treatment. Ten μg/ml mouse IgG was used as an isotype control to anti-leptin antibody. (D) Immunoblot analysis showing the expression of claudin-5, occludin, VE-cadherin, and ZO-1 in HDLECs treated with leptin (10, 100 ng/ml) for 24 h. β-actin was used as a loading control. Histograms represent relative expression of claudin-5, occludin, VE-cadherin, and ZO-1 proteins in HDLECs exposed to 10 or 100 ng/ml leptin for 24 h as determined by densitometry analysis. Results are expressed as means ± SEM of four independent experiments; t-test. n.s.: not significant.