Correction: Chen et al. Heteronemin Suppresses Lymphangiogenesis Through ARF-1 and MMP-9/VE-Cadherin/Vimentin. Biomedicines 2021, 9, 1109

Abstract

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Figure 1

Heteronemin is a VEGFR-3 binding compound in human lymphatic endothelial cells. (A) The structure of heteronemin (indicated as SP-1). (B) Docking models of SP-1-targeted VEGFR-3. The structure of VEGFR-3 was downloaded from PDB (accession: 4BSJ_A) and represented as gray. SP-1, drawn by ChemDraw Ultra 9.0, rendered in the representation of green stick. Close-up of SP-1 docking site (best energy mode) was prepared using Discovery Studio 4.1. (C) Human lymphatic endothelial cells (LECs) were treated with SP-1. Then, the expression of VEGFR-3 and phospho-VEGFR-3 (p-VEGFR-3) was determined by Western blot analysis. The quantitative densitometry of the relative levels of VEGFR-3 and phospho-VEGFR-3 was measured by Image-Pro Plus. (D) Cells were stimulated with SP-1; the phospho-VEGFR-3 protein expression in cell lysate was measured by ELISA. A representation experiment is shown as the mean ± S.D. for three wells (* p < 0.05). Similar results were observed from three independent experiments.

Figure 3

Heteronemin impairs the in vivo VEGF-C-stimulated lymphangiogenesis in mouse ear collagen sponges. Gelatin sponges were soaked with either medium containing VEGF-C (1 μg/mL) as a positive control or VEGF-C combined with the indicated concentration of heteronemin (represent as ‘SP-1’ in the graphs). Sponges were implanted between the two skin layers of mice ears for 3 weeks. (A) Lymphatic vasculatures were examined by LYVE-1 immunostainings. (bars = 100 and 70 μmon higher magnification). (B) The graphs represent the computerized quantification of the densities of lymphatic vessels, defined as the area occupied by vessels divided by the area of the sponge section. The data are expressed as the means ± SEM of five mice. * p < 0.05 versus medium sponges; # p < 0.05 versus VEGF-C-stimulated sponges.

Figure 4

Heteronemin inhibited cell growth and arrested the distribution of cell cycle in human lymphatic endothelial cells. (A) LECs were incubated with various doses (0–100 μM) of heteronemin (indicated as SP-1) in a 96-well plate for 24 h. Cell viability was determined by MTT assay after treatment. The maximal non-toxic dose was chosen for further experiments. (B) Cells were treated with SP-1 for 8 h; then, the cytotoxicity was determined using LDH assay. (C) Cells were treated with various concentrations of SP-1 for 24 h and were then analyzed using flow cytometry. Histograms represent the percentage of cells in each cell cycle phase. The graph corresponds to the distribution of cell subpopulation percentages expressed as means ± SEM of five independent assays. * p < 0.05 compared with solvent control (0.01% DMSO).

Figure 5

Heteronemin inhibited mitogen-activated protein kinase and transcription factors in human lymphatic endothelial cells. Cells were treated with the indicated concentrations of heteronemin (represented as SP-1); subsequently, the indicated (A) MEK/ERK and (C) p65 phosphorylated and total proteins were determined by Western blot analysis. The quantitative densitometry of phosphorylated (B) MEK/ERK and (D) p65 protein was performed with Image-Pro Plus. Data are expressed as mean ± SEM of five independent experiments. * p < 0.05, compared with the control group.

Figure 6

Heteronemin suppressed the tube formation, migration and phosphorylation of VEGF-C/VEGFR-3/LYVE-1 in human lymphatic endothelial cells. (A) Cells were treated with the indicated concentrations of heteronemin (indicated as SP-1). The capillary-like structure formation was examined by tube formation (scale bar = 500 μm; 20× magnification). (B) The quantification of tube formation was performed using Image-J to validate the anti-lymphangiogenic property of SP-1. (C) LECs were treated with SP-1. Then, the expression of LYVE-1and PROX-1 was determined by Western blot analysis. (D) The quantitative densitometry of the relative levels of LYVE-1 and PROX-1 was measured by Image-Pro Plus. Data are expressed as the mean ± SEM of at least three independent experiments. * p < 0.05; ^# p < 0.01 compared with the control group.