Novel application assigned to toluquinol: inhibition of lymphangiogenesis by interfering with VEGF-C/VEGFR-3 signalling pathway

Abstract

Background and purpose: Lymphangiogenesis is an important biological process associated with the pathogenesis of several diseases, including metastatic dissemination, graft rejection, lymphoedema and other inflammatory disorders. The development of new drugs that block lymphangiogenesis has become a promising therapeutic strategy. In this study, we investigated the ability of toluquinol, a 2-methyl-hydroquinone isolated from the culture broth of the marine fungus Penicillium sp. HL-85-ALS5-R004, to inhibit lymphangiogenesis in vitro, ex vivo and in vivo.

Experimental approach: We used human lymphatic endothelial cells (LECs) to analyse the effect of toluquinol in 2D and 3D in vitro cultures and in the ex vivo mouse lymphatic ring assay. For in vivo approaches, the transgenic Fli1:eGFPy1 zebrafish, mouse ear sponges and cornea models were used. Western blotting and apoptosis analyses were carried out to search for drug targets.

Key results: Toluquinol inhibited LEC proliferation, migration, tubulogenesis and sprouting of new lymphatic vessels. Furthermore, toluquinol induced apoptosis of LECs after 14 h of treatment in vitro, blocked the development of the thoracic duct in zebrafish and reduced the VEGF-C-induced lymphatic vessel formation and corneal neovascularization in mice. Mechanistically, we demonstrated that this drug attenuates VEGF-C-induced VEGFR-3 phosphorylation in a dose-dependent manner and suppresses the phosphorylation of Akt and ERK1/2.

Conclusions and implications: Based on these findings, we propose toluquinol as a new candidate with pharmacological potential for the treatment of lymphangiogenesis-related pathologies. Notably, its ability to suppress corneal neovascularization paves the way for applications in vascular ocular pathologies.

Figure 1

Toluquinol inhibits LEC proliferation and affects LEC viability. (A) Chemical structure of toluquinol. (B) Representative curve with the dose‐dependent effect of toluquinol on the in vitro growth of LECs. Cell proliferation is represented as a percentage of untreated cells. Each point represents the mean of quadruplicates; SD values were typically lower than 10% of the mean values and are omitted for clarity. The IC₅₀ value was calculated from dose–response curves as the concentration of compound yielding 50% of control cell survival. It is expressed as means ± SEM of five independent experiments. (C) Percentage of cell viability and cell toxicity after toluquinol treatment for 14, 24, 48 and 72 h. Results are expressed as the percentage of control (untreated cells) viability and toxicity (mean ± SEM of five independent experiments). Controls (100% of viability and 0% cytotoxicity) are not presented for each time point for clarity. Mann–Whitney–Wilcoxon test was used to decide if the differences among control (untreated cells) and toluquinol‐treated cells were statistically significant. ^* P < 0.05 versus control.

Figure 2

Toluquinol inhibits LEC migration. Confluent LEC monolayers were wounded, and fresh culture medium was added in either the absence or presence of the indicated concentrations of toluquinol. Photographs were taken at the beginning of the assay and after 24 and 48 h of incubation. All assays were performed in the presence of mitomycin C to avoid a proliferative effect on LEC. (A) Representative pictures of cell migration. Black drawings in pictures delineate the initial (time 0) wound edges (bar = 100 μm). (B) Quantification of LEC migration. Results are expressed as the percentage of the initial wounded area recovered by endothelial cells after 24 or 48 h (mean ± SEM of five independent experiments). Mann–Whitney–Wilcoxon test was the test used to analyse if the differences among control (untreated cells) and toluquinol‐treated cells were statistically significant. ^* P < 0.05 versus control.

Figure 3

Toluquinol suppresses LEC tubulogenesis. LECs were embedded in a collagen gel, maintained in culture for 24 h in order to form tubes and photographed under a phase contrast microscope. Toluquinol inhibited LEC tubulogenesis in a dose‐dependent manner at non‐toxic doses. (A) Representative pictures at different toluquinol concentrations (bar = 200 μm). (B) Quantification of the density of the tube area. Values are expressed as mean ± SEM of five independent assays, and Mann–Whitney–Wilcoxon test was used to evaluate statistically significant differences between control (untreated cells) and toluquinol‐treated cells. ^* P < 0.05 versus control.

Figure 4

Toluquinol inhibits LEC sprouting from spheroids. LEC spheroids embedded in a collagen‐methyl cellulose gel extended protrusion sensing the environment and formed tube‐like structures. Spheroids were treated or not (control) with toluquinol. (A) Cell sprouting is inhibited by increasing concentrations of toluquinol (bar = 100 μm). (B) Graphs correspond to the quantification of (i) the convex envelope area (area of minimal convex polygon containing the spheroid core as well as the migrated cells) (left panel) and (ii) the area occupied by migrating cells (middle panel). The graph at right displays LEC density as a function of the distance to the spheroid centre. Values are expressed as mean ± SEM of five tests with at least eight spheroids quantified from each one. Mann–Whitney–Wilcoxon test was used to decide if the differences among control (untreated spheroids) and toluquinol‐treated spheroids were statistically significant. ^* P < 0.05 versus control. (C) Cells stained orange with CellTracker Orange CMRA Dye and cells stained green with CellTracker Green CMFDA Dye were mixed in a 1:1 proportion before generating spheroids. After 12 h, cell sprouted from spheroids (1: non‐treated cells). The treatment of mixed spheroids (green + orange cells) with toluquinol during the migration assay led to a blockade of cell migration (2: toluquinol‐treated cells). When LECs were treated with toluquinol (5 μM) for 24 h (pretreated LECs), washed with PBS (toluquinol elimination), stained in green, mixed with orange untreated cells and treated without toluquinol during the migration assay (3: toluquinol‐pretreated cells), migration occurred in a similar way as in control conditions, revealing the reversibility of the inhibition induced by toluquinol (bar = 100 and 50 μm on higher magnification).

Figure 5

Toluquinol induces LEC apoptosis after 14 h of treatment. LECs were cultured in the presence of different toluquinol concentrations for 14 h, and then apoptosis was evaluated by Hoechst staining, flow cytometry and caspase‐3/‐7 activity. (A) Representative pictures showing the effect of toluquinol on nuclei morphology after Hoechst staining. The white circles delineate the chromatin condensation of apoptotic nuclei (bar = 100 μm). (B) Percentage of cells showing chromatin condensation per field. Values are expressed as mean ± SEM of the counts evaluated in 10 fields from five independent experiments (chromatin‐condensed cells or apoptotic cells were counted by fluorescence microscopy; total cells were counted by bright field microscopy). (C) Representative histograms showing the effect of toluquinol on LEC cycle distribution. After incubation with toluquinol, cells were stained with propidium iodide, and percentages of subG1, G1 and S/G2/M subpopulations were determined using a MoFlo DakoCytomation cytometer. (D) The graph corresponds to the distribution of cell subpopulation percentages expressed as means ± SEM of five independent assays. (E) Effect of toluquinol treatment for 14 h on LEC caspase‐3/‐7 activity. Results are expressed as mean ± SEM of five independent assays. (F) Effect of toluquinol 5 μM treatment on LEC caspase‐3/‐7 activity over time. Results are expressed as mean ± SEM of five independent assays. Controls (caspase‐3/‐7 activity = 100) are omitted for clarity. Statistically significant differences between control (untreated cells) and toluquinol‐treated cells were determined with the Mann–Whitney–Wilcoxon test. ^* P < 0.05 versus control.

Figure 6

Toluquinol suppresses VEGFR‐3 phosphorylation and downstream signalling targets. LECs were cultured in serum‐depleted conditions for 24 h, and incubated or not with different toluquinol concentrations for 2 h and then stimulated for 30 min with VEGF‐C (A), VEGF‐C156S (B) or VEGF‐A (C). Cell lysates were collected, and western blot analyses were performed. (A) Representative western blots showing the effects of toluquinol treatment on the phosphorylated VEGFR‐3, Akt and ERK1/2 in LECs stimulated with VEGF‐C (400 ng·mL⁻¹). (B) Illustrative western blots showing the impact of toluquinol treatment on VEGFR‐3, Akt and ERK1/2 phosphorylations in LECs stimulated with VEGF‐C156S (500 ng·mL⁻¹). (C) Representative western blots showing the interference of toluquinol treatment on VEGFR‐2, Akt and ERK1/2 phosphorylations in LECs stimulated with VEGF‐A (100 ng·mL⁻¹).

Figure 7

Toluquinol inhibits lymphatic outgrowth in mouse lymphatic ring assay. Mouse lymphatic duct explants embedded in type I collagen gel were cultured in the absence (control) or presence of different doses of toluquinol for 7 days under hypoxic conditions. (A) Representative micrographs of lymphatic rings (bar = 500 μm). (B) For quantification, binarized images from 10 rings per condition were subjected to a grid corresponding to successive increments at fixed intervals of thoracic duct boundary, and the number of microvessel grid intersections (Ni) at day 7 of incubation was calculated. Values are expressed as means ± SEM of 10 different rings. The graph (at right) corresponds to LEC density at a distance (d) = 0.25 mm to the ring border. Mann–Whitney–Wilcoxon test was used to determine if the differences among control (untreated rings) and rings incubated with toluquinol were statistically significant. ^* P < 0.05 versus control.

Figure 8

Toluquinol blocks thoracic duct (TD) development in zebrafish model. Transgenic Fli1:eGFPy1 zebrafish embryos were incubated in zebrafish water with the indicated concentrations of toluquinol at 28.5°C for 4 days, and then, thoracic duct length was analysed in the anaesthetized embryos. (A) Schematic drawing of a 5 dpf zebrafish embryo with the main anatomical features. Abbreviations used on this drawing denote as follows: dorsal longitudinal anastomotic vessels (DLAV), thoracic duct (TD), dorsal artery (DA), posterior cardinal vein (PCV), parachordal line (PAC), intersegmental vessels (ISV), arterial intersegmental vessels (aISV), venous intersegmental vessels (vISV) and intersegmental lymphatic vessels (ISLV). (B) Representative pictures of untreated and treated zebrafishes are shown. Zebrafish thoracic duct is indicated with white arrowheads on lower magnification pictures, and it is included in white rectangles on higher magnification pictures. Abbreviations indicate the main zebrafish embryo anatomical structures (bar = 100 and 70 μm on higher magnification). (C) Quantification of the defective thoracic duct formation at 5 dpf determined by the percentages of embryos with severe, drastic, moderate and no lymphatic defects, expressed as % of thoracic duct length. A total of 50 embryos were analysed in each experimental condition.

Figure 9

Toluquinol impairs the in vivo VEGF‐C‐stimulated lymphangiogenesis in mouse ear collagen sponges. Gelatin sponges were soaked with either DMEM containing the vehicle (DMSO) as negative control or VEGF‐C (1 μg·mL⁻¹) as a positive control or VEGF‐C and different concentrations of toluquinol, (indicated with ‘T’ in the graphs). Sponges were implanted between the two skin's layers of mice ears for 3 weeks. (A) Lymphatic and blood vasculatures were examined by LYVE‐1 (green) and CD31 (red) immunostainings respectively. DAPI staining was used to detect cell nuclei (blue) (bars = 1500 and 500 μm on higher magnification). (B) The graphs represent the computerized quantification of the densities of lymphatic (left panel) or blood (middle panel) vessels, defined as the area occupied by vessels divided by the area of sponge section. Data are expressed as means ± SEM of five mice. The right graph corresponds to vessel distribution from the sponge edge to its centre. The arrows indicated the maximal distance of LEC migration (Lmax). Mann–Whitney–Wilcoxon test was used to evaluate statistically significant differences between sponges soaked with VEGF‐C + toluquinol and control sponges with VEGF‐C. ^* P < 0.05 versus VEGF‐C‐stimulated sponges.

Figure 10

Toluquinol reduces corneal neovascularization in cauterized mouse corneas. Corneal lymph/angiogenesis was induced by thermal cauterization, and mice were administered 75 nmol toluquinol i.p. daily (treated mice) or PBS (control mice). Corneas were flat mounted at day 9 post‐injury and immunostained for detecting lymphatic vessels (LYVE‐1 positive, in green) and blood vessels (CD31 positive, in red). Whole‐mounted corneas were observed under a fluorescent (A–D) or a confocal (E–F) microscope. (A–D) Representative pictures of corneas from non‐treated (A, B) and treated (C, D) mice. Lymphatic vessels and blood vessels appear in green and in red respectively (bars = 1000 μm in A and C; 500 μm in B and C). (E–F) Representative pictures of filopodia‐like structures (white arrowheads) displayed by migrating LECs in corneas from control (E) and toluquinol‐treated mice (F) (bars = 10 μm). (G–H) Computer‐assisted quantification was based on the splitting of green and red channel to dissociate lymphatic from blood networks. The vessel area (area covered by neoformed vessels), total length (cumulative length of the vessels), branching (number of bifurcations) and end‐point (number of sprout tips) densities were calculated after image binarization. All results were divided by the total cornea area. Results are expressed as the mean ± SEM of 15 mice. (I) A grid was applied on each cornea picture to establish the distribution curves of capillaries around the limbal vessels, and it is presented as the number of intersections (Ni) versus the distance to the limbus. (J) Graphs represent the number (left) and length (right) of filopodia‐like structures in a total length of 25 μm at the end of the lymphatic vessel. For these quantifications, 10 pictures were quantified per mouse, and corneas from five mice were evaluated. Mann–Whitney–Wilcoxon test was the statistical test used to evaluate significant differences between control mice (PBS) and toluquinol‐injected mice. ^* P < 0.05 versus control mice.